(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property
Organization
International Bureau
iEIIIIISIIH I III' llllllll!ltlll[;il illlll IIIINEIIIMIIHtll IllllJil 1H! Ill
(43) International Publication Date (10) International Publication Number
27 January 2005 (27.01.2005) PCT WO 2005/007796 A2
(51) International Patent Classification 7 :
C12M
(21) International Application Number:
PCT/IL2004/000661
(22) International Filing Date: 20 July 2004 (20.07.2004)
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
60/488,408
60/488,409
60/517,073
60/517,084
60/544,356
60/544,357
PCT/IL04/000571
21 July 2003 (21 .07.2003) US
21 July 2003 (21.07.2003) US
5 November 2003 (05.11.2003) US
5 November 2003 (05.1 1 .2003) US
17 February 2004 (17.02.2004) US
17 February 2004 (17.02.2004) US
27 June 2004 (27.06.2004) IL
(71) Applicant (for all designated States except US): MOLEC-
ULAR CYTOMICS LTD* [CY/CY]; P.O. Box 21255,
1505 Nicosia (CY).
(72) Inventors; and
(75) Inventors/Applicants (for US only): DEUTSCH,
Mordechai [H7IL]; 73 Moshav Olesh, 42 855 Doar Na
Lev-HaSharon (IL). HERZBERG, Max [IL/IL]; Moshav
Sitria, P. O. Box 7, 76 834 Emeq Soreq (IL). TJLROSH,
Rcuvcn [IL/IL]; 20 Ben Gurion Street, 44257 Kfar Saba
(IL). DEUTSCH, Assaf [IL/IL]; 12 Moshav Tzofaria, 60
932 Moshav Tzofaria (IL).
(74) Agent: G.E. EHRLICH (1995) LTD.; 11 Menachem Be-
gin Street, 52 521 Ramat Gan (IL).
(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG,
PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM,
TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM,
ZW.
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
FR, GB, GR, HU, IE, IT, LU, MC, NL, PL, PT, RO, SE, SI,
SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
GW, ML, MR, NE, SN, TD, TG).
Published:
— without international search report and to be republished
upon receipt of that report
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
<
ON
(54) Title: IMPROVED MULTIWELL PLATE
^ (57) Abstract: A multiwell plate having a plurality of picowells on the bottom of the wells of the plate as well as methods of
Q producing the multiwell plate are provided. Provided is also a method of handling living cells by providing an ordered array of living
cells immobilized in a non-fluid matrix, contacting the living cells with a stimulus; and detecting a response to the stimulus. The
present invention is also of a method of producing an ordered array of living cells.
WO 2005/007796 PCT/EL2004/000661
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IMPROVED MULTIWELL PLATE
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to the field of cellular biology and more
particularly, to an improved device and method for the study of cells. Specifically, the
present invention is of an improved multiwell plate and methods for making the same
that allows the use of automatised sample handling methods for the study of single
living cells. Further, the present invention is of a device substantially being an ordered
array of living cells.
Combinatorial methods in chemistry, cellular biology and biochemistry are
essential for the near simultaneous preparation of multitudes of active entities such as
molecules. Once such a multitude of molecules is prepared, it is necessary to study the
effect of each one of the active entities on a living organism.
The study of the effects of stimuli, such as exposure to active entities, on living
organisms is preferably initially performed on living cells. Since, cell-functions include
many interrelated pathways, cycles and chemical reactions, the study of an aggregate of
cells, whether a homogenous or a heterogeneous aggregate, does not provide
sufficiently detailed or interpretable results: rather a comprehensive study of the
biological activity of an active entity may be advantageously performed by examining
the effect of the active entity on a single isolated living cells. Thus, the use of single-cell
assays is one of the most important tools for understanding biological systems and the
influence thereupon of various stimuli such as exposure to active entities.
The combinatorial preparation of a multitudes of active entities coupled with the
necessity of studying the effect of each one of the active entities on living organisms
using a single-cell assay, requires the development of high-throughput single live cell
assays.
In the art, various different methods for studying living cells are known.
Multiwell plates having 6, 12, 48, 96, 384 or even 1536 wells on a standard ca.
8.5 cm by ca. 12.5 cm footprint are well known in the art. Such multiwell plates are
provided with an 2n by 3n array of rectangular packed wells, n being an integer. The
diameter of the wells of a plate depends on the number of wells and is generally greater
than about 250 microns (for a 1536 well plate). The volume of the wells depends on the
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number of wells and the depth thereof but generally is greater than 5 x 10" 6 liter (for a
1536 well plate).
Multiwell plates are commercially available from many different suppliers.
Multiwell plates made from many different materials are available, including but not
5 limited to glass, plastics, quartz and silicon. Multiwell plates having wells where the
inside surface is coated with various materials, such as active entities, are known.
The standardization of the formats of multiwell plates is a great advantage for
researchers as the standardization allows the production of standardized products
including robotic handling devices, automated sample handlers, sample dispensers, plate
10 readers, observation devices, plate washers, software and such accessories as
multifilters.
Although exceptionally useful for the study of large groups of cells, multiwell
plates are not suitable for the study of individual cells or even small groups of cells due
to the large, relative to the cellular scale, size of the wells. Cells held in such wells
15 either float about a solution or adhere to a well surface. When cells float about in a well,
specific individual cells are not easily found for observation. When cells adhere to a
well surface, the cells adhere to any location in the well, including anywhere on the
bottom of the well and on the walls of the well. Such variability in location makes high-
throughput imaging (for example for morphological studies) challenging as acquiring an
20 individual cell and focusing thereon is extremely difficult. Such variability in location
also makes high-throughput signal processing (for example, detection of light emitted
by a single cell through fluorescent processes) challenging as light must be gathered
from the entire area of the well, decreasing the signal to noise ratio. Further, a cell held
in a well of a multiwell plate well can be physically or chemically manipulated (for
25 example, isolation or movement of a single selected cell or single type of cell, changing
media or introducing drugs) only with difficulty. Further, the density of cells held singly
in the wells of a multiwell plate is very low (about 1536 cells in 65 cm 2 , or 24 cells cm*
2 ) Thus, multiwell plates are in general only suitable for the study of homogenous or
heterogenous aggregates of cells as a group.
30 An additional disadvantage of multiwell plates is during the study of cells
undergoing apoptosis. One method of studying cells is by exposing cells in a monolayer
of cells adhered to the bottom of the well of a multiwell plate to a stimulus. As is known
WO 2005/007796 PCT/EL2004/000661
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to one skilled in the art, one of the most important processes that a cell potentially
undergoes is apoptosis and it is highly desirable to observe a cell throughout the
apoptosis process. However, once a cell begins the apoptosis process, the adhesion of
the cell to the bottom of the well is no longer sufficient: the cell detaches from the
5 bottom and is carried away by incidental fluid currents in the well. The cell is no longer
observable and its identity lost.
In the art, a number of method and devices have been developed for the study of
individual cells or a small number of cells as a group. Many such methods are based on
using picowell-bearing devices. A picowell-bearing device is a device for the study of
10 cells that has at least one picowell-bearing component for study of cells. A picowell-
bearing component is a component having at least one, but generally a plurality of
picowells, each picowell configured to hold at least one cell. The term "picowell" is
general and includes such features as dimples, depressions, tubes and enclosures. Since
cells range in size from about 1 microns to about 100 (or even more) microns diameter
15 there is no single picowell size that is appropriate for holding a single cell of any type.
That said, the dimensions of the typical individual picowell in the picowell-bearing
components known in the art have dimensions of between about 1 microns up to about
200 microns, depending on the exact implementation. For example, a device designed
for the study of single isolated 20 micron cells typically has picowells of dimensions of
20 about 20 microns. In other cases, larger picowells are used to study the interactions of a
few cells held together in one picowell. For example, a 200 micron picowell is
recognized as being useful for the study of the interactions of two or three cells, see
PCT patent application IL01/00992 published as WO 03/035824.
One feature that increases the utility of a picowell-bearing device is that each
25 individual picowell is individually addressable. By individual addressability is meant
that each picowell can be registered, found or studied without continuous observation.
For example, while cells are held in picowells of a picowell-bearing component, each
cell is characterized and the respective picowell where each cell is held is noted. When
desired, the observation component of the picowell-bearing device is directed to the
30 location of the picowell where a specific cell is held. One method of implementing
individual addressability is by the use of fiducial points or other features (such as signs
or labels), generally on the picowell-bearing component. Another method of
WO 2005/007796
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implementing individual addressability is by arranging the picowells in a picowell-array
and finding a specific desired picowell by counting. Another method of implementing
individual addressability is by providing a dedicated observation component for each
picowell.
5 In the art, the picowell-bearing component of picowell-bearing devices is often a
chip, a plate or other substantially planar component. Herein such a component is
termed a "carrier". In the art, there also exist non-carrier picowell-bearing components
of picowell-bearing devices, for example, bundles of fibers or bundles of tubes.
Mrksich and Whitesides, Ann. Rev. Biophys. Biomol Struct. 1996, 25, 55-78;
10 Craighead et aL, J. Vac. Sci. TechnoL 1982, 20, 316; Singhvi et al % Science 1994, 264,
696-698; Aplin and Hughes, AnalyU Biochem. 1981, 113, 144-148 and U.S. Patent
5,324,591 all teach of devices including arrays of spots of cell-attracting or cell-binding
entities on a plate. In such devices, the spots serve as picowells, binding to cells through
a variety of chemical bonds. In such devices, the plate is the picowell-bearing
15 component of the device. Due to the size of the spots, each such picowell generally
holds more than one cell. To reduce interaction between cells held at different
picowells, the spots must be spaced relatively far apart, reducing loading as expressed in
terms of picowells per unit area. Even with generous spacing, in such picowell-bearing
components held cells are not entirely isolated from mutual interaction, nor can cells be
20 subject to individual manipulation. The fact that the cells are not free-floating but are
bound to the plate through some interaction necessarily compromises the results of
experiments performed.
In U.S. Patent 6,103,479, the picowell-bearing component is a transparent
carrier provided with a non-uniform array of picowells, each well functionalized with
25 chemical entities that bind to cells specifically or non-specifically. Each picowell is of
approximately 200 to 1000 micron diameter and is configured to hold a plurality of
cells. The inter picowell areas are hydrophobic so as not to attract cells. In addition to
the carrier, a device of U.S. Patent 6,103,479 is provided with a glass, plastic or silicon
chamber-bearing plate in which individually addressable microfluidic channels are
30 etched that mates with the carrier. When brought together, the carrier and chamber-
bearing plate constitute a cassette in which each cell is bound to the carrier and isolated
in a chamber provided with an individual fluid delivery system. Reagents are provided
WO 2005/007796
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through the fluid delivery system and observed by the detection of fluorescence. In
order to provide space for the walls of the chambers, the inter picowell areas of the
carrier are relatively large, reducing loading as expressed in terms of picowells per unit
area. Subsequent to study, the cassette is separated into the two parts and the micro-
5 patterned array of cells processed further. In some embodiments, the chamber-bearing
plate is made of polytetrafluoroethylene, polydimethylsiloxane or an elastomer. As held
cells do not make contact with the chamber-bearing plate it is not clear what advantages
are to be had when providing a chamber-bearing plate of such esoteric materials.
In U.S. Patent 4,729,949, a device is taught for trapping individual cells in a
10 picowell-bearing carrier, the carrier being substantially a plate having a plurality of
picowells that are individually-addressable tapered apertures of a size to hold individual
cells. Suction applied from the bottom surface of the plate where the picowells are
narrow creates a force that draws cells suspended in a fluid above the carrier into the
wide end of the picowells on the surface of the carrier to be held therein. Using the
1 5 teachings of U.S. Patent 4,729,949 a specific group of cells (having dimensions similar
to that of the wide end of the picowells) can be selected from amongst a group of cells
and held in the carrier. Although the cells are subjected to common stimuli, the fact that
the picowells are individually addressable allows the effect of a stimulus on an
individual cell to be observed. A carrier of U.S. Patent 4,729,949, is generally made of
20 metal such as nickel and prepared using standard photoresist and electroplating
techniques. In a carrier of U.S. Patent 4,729,949, the inter picowell areas of the carrier
are relatively large, leading to a low loading as expressed in terms of picowells per unit
area. Further, the suction required to hold cells in picowells of a carrier of U.S. Patent
4,729,949 deforms held cells and makes a significant portion of the cell membranes
25 unavailable for contact, both factors that potentially compromise experimental results.
Study of cells with non-fluorescence based methods generally gives poor results due to
reflections of light from the carrier.
In PCT patent application US99/04473 published as WO 99/45357 is taught a
picowell-bearing device produced by etching the ends of a bundle of optical fibers
30 (apparently of glass) while leaving the cladding intact to form a picowell-bearing
component that is a bundle of tubes. The size of the hexagonal picowells is
demonstrated to be as small as 7 micron wide, 5 micron deep and having a volume of
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1.45 x 10" u liter. The inter picowell area is quite large due to the thickness of the
cladding of the optical fibers. Cells held in each picowell are independently observable
through a respective optical fiber. In some embodiments, the inside surface of the
picowells is coated with a film of materials such as collagen, fibronectin, polylysine,
5 polyethylene glycol, polystyrene, fluorophores, chromophores, dyes or a metal. Loading
the picowell-bearing component of PCT patent application US99/04473 includes
dipping the optical fiber bundle in a cell suspension so that cells adhere to the picowells.
There are a number of disadvantages to the teachings of PCT patent application
US99/04473. The fact that the cells are studied only subsequent to adhesion to the
10 picowells necessarily influences the results of experiments performed. Since cell
proliferation generally begins soon after adhesion, it is never clear if a signal detected
results from a single cell or a plurality of cells. It is is not clear where exactly in a
picowell a cell is held and therefore what percentage of light emitted from a cell travels
to a detector. The fact that emitted light travels through an optical fiber leads to loss of
1 5 time-dependent and phase information.
In unpublished copending PCT patent application IL04/00192 of the Applicant
filed 27 June 2004 is taught a picowell-bearing device produced by bundling together
glass capillaries, each glass capillary attached to an independent fluid flow generator
such as a pump. A cell held in a first picowell is transferred to a second picowell by the
20 simultaneous application of an outwards flow from the first picowell and an inwards
flow into the second picowell.
A preferred device for the study of cells is described in PCT patent application
IL01/00992 published as WO 03/035824. The device 10, depicted in Figure 1, is
provided with a transparent carrier 12 as a picowell-bearing component. Carrier 12 is
25 substantially a sheet of transparent material (such as glass or polystyrene) on the surface
of which features such as inlet connectors 14, fluid channels 16, picowells (in Figure 1 a
well-array 18), a fluid reservoir 20 and an outlet connector 22. Carrier 12 is immovably
held in a holder 24 having a cutout window of a size and shape to accept carrier 12.
Other components of device 10 not depicted include flow generators, observation
30 components, external tubing and the like. When a cover slip (not depicted) is placed or
integrally formed with carrier 12, fluid channels 16, picowell-array 18 and reservoir 20
are sealed forming channels that allow transport of fluids and reagents to cells held in
"1
t
WO 2005/007796 PCT/IL2004/000661
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picowell-array 18. The picowells are configured to hold a predetermined number of
cells (one or more) of a certain size and are preferably individually addressable both for
examination and manipulation.
Figure 2 is a reproduction of a photograph of a different carrier 26 held in a
5 holder 24. A first syringe 28 as an inlet flow generator is in communication with an inlet
connector 14 by a capillary tube 30. Inlet connector 14 is in communication with
picowell-array 18 through a fluid passage 16. Picowell-array 18 is in communication
with outlet connector 22 through a fluid passage 16. A second syringe 32 as an outlet
flow generator is in communication with outlet connector 22 through capillary tube 34.
10 PCT patent application IL01/00992 also teaches methods of physically
manipulating cells held in a picowell-bearing device using, for example, individually
addressable microelectrodes (found in the picowells or in the cover slip) or optical
tweezers. Typical physical manipulations include moving cells into or out of picowells.
One useful method that is implemented using a device of PCT patent application
15 IL0 1/00992 is that cells, each held alone in a respective picowell, are examined (either
in the presence or absence of reagents) and based on the results of the examination, cells
with a certain characteristic are selected to remain in a respective picowell while cells
without the certain characteristic are removed from a respective picowell and ejected by
the application of a flow in parallel to the surface of the carrier, generated by a flow
20 generator.
An additional feature of the teachings of PCT patent application IL01/00992 is
that, in some embodiments, the picowells are juxtaposed, that is, the area occupied by a
picowell-array is substantially entirely made up of picowells with little or no inter
picowell area, see Figure 3. Figure 3 is a reproduction of a photograph of part of a
25 picowell-array 18 from the top of a carrier 12 of PCT patent application IL01/00992. In
Figure 3 is seen a plurality of hexagonal picowells 36, some populated with living cells
38. It is seen that the inter picowell areas 40 make up only a minor percentage of the
total area of picowell-array 18. This feature allows near tissue-density packing of cells,
especially in single-cell picowell configurations. For example, a typical device of PCT
30 patent application IL01/00992 having a 2 mm by 2 mm picowell-array of hexagonally-
packed juxtaposed picowells of 10 micron diameter and no inter picowell area includes
about 61600 picowells. This feature also allows simple picowell loading: a fluid
WO 2005/007796
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containing suspended cells is introduced in the volume above the picowells. Since there
is little inter picowell area, cells settle in the picowells.
Despite the utility of the device taught in PCT patent applications ILO 1/00992,
the use of the device is too labor intensive for certain high-throughput implementations.
5 Amongst other reasons the large amount of labor is required because there exist no
commercially available robotic systems optimized for use with the devices.
It would be highly advantageous to have a device for the study of cells not
having at least some of the disadvantages of the prior art.
10 SUMMARY OF THE INVENTION
The present invention successfully addresses at least some of the shortcomings
of the prior art by providing an improved multiwell plate and a new device, a method
for producing the improved multiwell plate and the new device and new methods for
handling living cells.
15 According to the teachings of the present invention there is provided a multiwell
plate comprising a plurality of wells wherein at the bottom surface of at least one well
of the plurality of wells is a plurality of picowells. Preferably, a plate of the present
invention has a footprint of a standard multiwell plate. Preferably, the plurality of wells
of a plate of the present invention comprises 6n wells arranged in a 2n by 3n array,
20 where n is an integer greater than 0, the wells preferably being arranged in rectangular
packing. Preferred pluralities of wells are the commonly known pluralities of well such
as 6, 24, 96, 384 and 1536 wells. Most preferred are plates of 96 wells and 384 wells as
these formats are most popular and have many available accessories including fluid-
handling accessories such as fluid-handling robots.
25 In an embodiment of the present invention, the plurality of picowells comprises
individually addressable picowells. In an embodiment of the present invention, the
bottoms of all picowells in a given well of a plate of the present invention are
substantially coplanar. In an embodiment of the present invention, the bottoms of all
picowells of a plate of the present invention are substantially coplanar.
30 In an embodiment of the present invention, the picowells of a plurality of
picowells in a given well are juxtaposed. By juxtaposed is meant that in an area where
picowells are found, most of the area is picowell area and little of the area is inter
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picx)\vell area. According to a feature of the present invention, by juxtaposed is meant
that the inter picowell area between two picowells is less than or equal to 0.35, 0.25,
0.15, 0.10 or even 0.06 of the sum of the areas of the two picowells. In certain
embodiments of the present invention it is preferred that the inter picowell area be
5 substantially zero, that is that the rims of picowells are substantially knife-edged.
The dimensions of picowells of a multiwell plate of the present invention,
depending on the specific embodiment, are less than about 200 microns, less than about
100 microns, less than about 50 microns, less than about 25 microns or even less than
about 10 microns. In an embodiment of the present invention, picowells are configured
10 to hold no more than one living cell of a certain size at any one time. In an embodiment
of the present invention, picowells are configured to hold no more than a predetermined
number of living cells of a certain size at any one time.
In an embodiment of the present invention, the picowells are enclosures of
dimensions such that substantially at least one entire cell of a certain size is containable
1 5 within such an enclosure, each enclosure having an opening at the surface of the carrier,
the opening defined by a first cross section of a size allowing passage of a cell of the
certain size. Depending on the embodiment, the volume of such an enclosure is
typically less than about 1 x 10' M liter, less than about 1 x 10" i2 liter, less than about 1 x
10" 13 liter, less than about 1 x 10" 14 liter or even less than about 1 x 10* 15 liter.
20 Depending on the embodiment, the area of the first cross section of such an enclosure is
typically less than about 40000 micron 2 , less than about 10000 micron 2 , less than about
2500 micron 2 , less than about 625 micron 2 or even less than about 100 micron 2 . In an
embodiment of the present invention, picowells enclosures are configured to hold no
more than one living cell of a certain size at any one time. In an embodiment of the
25 present invention, picowells enclosures are configured to hold no more than a
predetermined number of living cells of a certain size at any one time.
In an embodiment of the present invention, the plurality of picowells comprises
picowells, wherein all picowells of the plate are substantially identical in size.
In another embodiment of the present invention, a first well of a plate of the
30 present invention includes a first plurality of picowells and a second well of a plate
includes a second plurality of picowells, wherein the first plurality of picowells and the
second plurality of picowells are substantially different. For example, in an embodiment
WO 2005/007796 PCT/IL2004/000661
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of the present invention the size of the picowells of the first plurality is substantially
different from the size of picowells of the second plurality of picowells.
A multiwell plate of the present invention is made of any suitable material.
Suitable materials include but are not limited to ceramics, elastomers, epoxies, glasses,
5 glass-ceramics, metals, plastics, polycarbonates, poiydimethylsiloxane, polyurethane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate, polystyrene,
polyvinyl chloride, rubber, silicon, silicon oxide and silicon rubber.
In an embodiment of the present invention, the bottom surface of the wells is
made of any suitable material. Suitable materials include but are not limited to ceramics,
10 elastomers, epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates,
poiydimethylsiloxane, polyethylenterephtalate glycol, polymers, polymethyl
methacrylate, polystyrene, polyurethane, polyvinyl chloride, rubber, silicon, silicon
oxide and silicon rubber.
In embodiments of the present invention, an entire plate of the present invention
15 and all components thereof are made of one material. In other embodiments, a plate of
the present invention is made up of a number of different materials, for example, as a
plurality of layers or as a coated structure.
In an embodiment of the present invention, the walls of wells of the plurality of
wells are integrally formed with the bottom surface of the wells.
20 In other embodiments, a plate of the present invention comprises at least one
distinct well-wall component attached to the bottom surface. Such a distinct well-wall
component is made of any suitable material. Suitable materials include but are not
limited to ceramics, elastomers, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, poiydimethylsiloxane, polyethylenterephtalate glycol, polymers,
25 polyurethane, polymethyl methacrylate, polystyrene, polyvinyl chloride, rubber, silicon,
silicon oxide and silicon rubber.
In an embodiment of the present invention, a plurality of picowells are integrally
formed with the bottom surface.
In an embodiment of the present invention, a plate of the present invention
30 comprises at least one distinct picowell-bearing component bearing a plurality of
picowells, the component attached to the bottom surface of a respective well or simply
resting within a respective well.
WO 2005/007796 PCT/IL2004/000661
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A suitable distinct picowell-bearing component is a carrier comprising a
plurality of picowells disposed on a surface, such as a carrier described in PCT patent
application IL01/00992 or in unpublished copending PCT patent application
IL04/00571 of the Applicant filed 27 June 2004 {vide infra). Picowell-bearing
5 components are made of any suitable material, including reversibly deformable
materials and irreversibly deformable materials. Suitable materials include but are not
limited to gels, hydrogels, waxes, hydrocarbon waxes, crystalline waxes, paraffins,
ceramics, elastomers, epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates,
polydimethylsiloxane, polyethylenterephtalate glycol, polymers, polymethyl
10 methacrylate, polystyrene, polyurethane, polyvinyl chloride, rubber, silicon, silicon
oxide and silicon rubber.
In an embodiment of the multiwell plate of the present invention, the picowell-
bearing component comprises a gel, preferably a transparent gel, preferably a hydrogel.
Gels suitable for use in making a picowell-bearing component of a plate of the
15 present invention include but are not limited to agar gels, agarose gels, gelatins, low
melting temperature agarose gels, alginate gels, room-temperature Ca 2+ -induced
alginate gels and polysaccharide gels. Depending on the embodiment, a suitable gel has
a water content of greater than about 80% by weight, greater than about 92% by weight,
greater than about 95% by weight, greater than about 97% by weight and even greater
20 than about 98% by weight. In an embodiment of the present invention, the gel includes
an active entity. Suitable active entities include, but are not limited to antibodies,
antigens, biological materials, chemical materials, chromatogenic compounds, drugs,
enzymes, fluorescent probes, immunogenes, indicators, ligands, nucleic acids, nutrients,
peptides, physiological media, proteins, receptors, selective toxins and toxins,
25 In an embodiment of the present invention, picowells have a bottom surface
made of a first material and borders, such as the borders delineating the picowells, made
of a second material, the second material being substantially different from the first
material In an embodiment of the present invention the first material is substantially the
material from which the bottom of the well is made, for example when the bottom
30 surface of the picowell is substantially the bottom surface of the well. In an embodiment
of the present invention, the second material is a fixed photoresist material.
WO 2005/007796 PCT/IL2004/000661
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In an embodiment of the plate of the present invention, the plurality of picowells
comprises picowells having an inside surface configured to delay proliferation of cells
held therein, for example, by delaying adhesion of living cells thereto. In an
embodiment of the plate of the present invention, the inside of a picowell comprises a
5 material that delays adhesion of living cells thereto, that is the picowell is substantially
fashioned from the adhesion-delaying material or the inside of the picowell is coated
with the adhesion-delaying material. A suitable material to coat the inside of a picowell
or from which to make a picowell comprises polydimethylsiloxane, is substantially
polydimethylsiloxane or is substantially pure polydimethylsiloxane.
10 In an embodiment of the present invention bottom surfaces of picowells making
up a plurality of picowells of a plate comprise a material having an index of refraction
similar to that of water. In a preferred embodiment of a plate of the present invention,
the index of refraction of the bottom surfaces is less than about 1 .4, less than about 1 .38,
less than about 1.36, less than about 1.35, less than about 1.34 or substantially equal to
15 that of water.
In an embodiment of the present invention, the plurality of picowells comprises
picowells having an inner surface coated with a layer of a material. Suitable materials
for coating an inner surface of a picowell of a plate of the present invention include but
are not limited to gels, hydrogels, polydimethylsiloxane, elastomers, polymerized para-
20 xylylene molecules, polymerized derivatives of para-xylylene molecules, rubber and ,
silicon rubber.
In an embodiment of the present invention, a plate of the present invention
further comprises a gel cover covering a plurality of the picowells, the cover made of a
gel; Suitable gels are as described hereinabove.
25 In an embodiment of the present invention, substantially the entire bottom
surface of a well is covered by a respective plurality of picowells.
In an embodiment of the present invention, a plate further comprises at least one
additional feature functionally associated with the plurality of picowells, especially
30 micro fluidic features. Suitable microfluidic features include but are not limited to
channels, coupling elements, drains, fluid channels, fluid reservoirs, input ports,
membranes, microreactors, microvalves, output ports, passages, plumbing routes,
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protruberances, pumps, transport channels and valves. Other suitable features include
but are not limited to light sources, magnetizable elements, optical components, optical
fibers, optical filters, protuberances, fiducial points and walls.
In an embodiment of the present invention, a plate further comprises a cover
5 slip, the cover slip and a plurality of picowells in a well configured so as to allow the
cover slip to rest above the plurality of picowells substantially in parallel to the bottom
surface of the well.
According to the teachings of the present invention, there is provided a method
of making a multiwell plate of the present invention, comprising: (a) contacting a
10 precursor material with a template including a negative of features of the plate so as to
create the features in the precursor material, the features including the plurality of
picowells; (b) fixing the features in the precursor material so as to fashion an incipient
plate; and (c) processing the incipient plate so as to fashion the multiwel plate of the
present invention.
15 Depending on the embodiment and the nature of the precursor material, fixing
includes such methods a heating the precursor material, cooling the precursor material,
polymerizing the precursor material, cross-linking the precursor material, curing the
precursor material, irradiating the precursor material, illuminating the precursor
material, gelling the precursor material, exposing the precursor material to a fixative and
20 waiting a period of time.
The template is generally made of a material that is rigid compared to the
precursor material. Suitable materials include but are not limited to reversibly
deformable materials, irreversibly deformable materials, ceramics, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane,
25 polyethylenterephtalate glycol, polymers, polymethyl methacrylate, paraffins,
polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon oxide, silicon rubbers
and wax.
Features created in the precursor material in addition to the plurality of picowells
include such features as channels, coupling elements, drains, fluid channels, fluid
30 reservoirs, input ports, light sources, magnetizable elements, membranes, microreactors,
microvalves, passages, optical components, optical fibers, optical filters, output ports,
plumbing routes, protruberances, pumps, transport channels, valves, walls and fiducial
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points. In an embodiment of the present invention, the features created in the precursor
material in addition to the plurality of picowells include the plurality of wells.
In an embodiment of the present invention, prior to contacting the template with
the precursor material, the precursor material is placed in a well of a preexisting
5 multiwell plate.
In an embodiment of the present invention, subsequent to the fixing of the
features, walls of the plurality of wells are attached to the incipient plate. Attaching
includes the use of methods employing adhesives or surface treatments such as plasma
treatments.
10 In an embodiment of the present invention the precursor material is an
irreversibly deformable material {vide infra) such as a wax, a paraffin, plastic or
polymer, and fixing the features simply includes separating the template from the
precursor material.
In an embodiment of the present invention the precursor material is a reversibly
15 deformable material {vide infra) such as a gellable fluid, a polymerizable material, a
powder, a fluid or a thermoplastic material.
In an embodiment of the present invention, the reversibly deformable precursor
material is a thermoplastic material at plastic temperature and fixing the features
includes cooling the thermoplastic material.
20 In an embodiment of the present invention, the reversibly deformable precursor
material is a polymerizable material and fixing the features includes polymerizing the
polymerizable material. Suitable polymerizable materials include but are not limited to
monomer solutions, crosslinkable polymers, vulcanizable polymers, polymerizable fluid
and thermosetting resins.
25 In a preferred embodiment, the polymerizable material is a polydimethylsiloxane
precursor mixture and fixing the features includes polymerizing the
polydimethylsiloxane precursor mixture so as to produce polydimethylsiloxane. In
another preferred embodiment, the polymerizable material includes urethane and fixing
the features includes polymerizing the urethane to produce polyurethane.
30 In an embodiment of the present invention, the reversibly deformable precursor
material is a gellable fluid and fixing the features includes gelling the gellable fluid.
Depending on the nature of the gellable fluid used, preferred methods of gelling the
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gellable fluid include of heating the gellable fluid, cooling the gellable fluid, irradiating
the gellable fluid, illuminating the gellable fluid, contacting the gellable fluid with a
gelling reagent and waiting a period of time for the gellable fluid to gel. Suitable
gellable fluids include but are not limited to agars, agaroses, gelatins, low melting
5 temperature agaroses, alginates, proteins, protein polysaccharides, room-temperature
Ca 2+ -inducable alginates and polysaccharides. A preferred gellable fluid is an alginate
solution where gelling the gellable fluid includes contacting the gellable fluid with a
gelling reagent, such as a gelling reagent including Ca 2+ ions. An additional preferred
gellable fluid is a low melting temperature agarose solution and gelling the gellable
1 0 fluid includes cooling the gellable fluid.
In an embodiment of the present invention, processing the incipient plate
comprises coating an inside surface of picowells of the plurality of picowells with a
layer of a coating material.
According to the teachings of the present invention there is provided an
15 additional method of making a multiwell plate of the present invention, comprising (a)
placing a photoresist material on a precursor plate; and (b) fixing a plurality of
picowells in the photoresist material. Preferably, the fixing of the plurality of picowells
comprises irradiating the photoresist material through a mask. A precursor plate is made
of a suitable material. Suitable materials include but are not limited to ceramics,
20 epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane,
polymers, polyethylenterephtalate glycol, polymethyl methacrylate, polystyrene,
polyurethanes, polyvinyl chloride, silicon and silicon oxide.
In an embodiment of the method of the present invention the precursor plate
comprises a multiwell plate. The photoresist material is placed in a well of the precursor
25 plate and the photoresist material irradiated inside the well.
In an embodiment of the present invention, subsequent to the fixing of the
features, walls of the plurality of wells are attached to the precursor plate. Attaching
includes the use of methods employing adhesives or surface treatments such as plasma
treatments.
30 In an embodiment of the present invention, subsequent to fixing the picowells in
the photoresist material, the inside surface of picowell of the plurality of picowells is
coated with a layer of a coating material.
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According to the teachings of the present invention, there is provided an
additional method for making a multiwell plate of the present invention by placing a
picowell-bearing component on a precursor plate. In a preferred embodiment, the
picowell-bearing component is attached to the precursor plate. Attaching includes the
5 use of methods employing adhesives or surface treatments such as plasma treatments. A
suitable picowell-bearing component includes a carrier comprising a plurality of
picowells disposed on a surface, such as a carrier described in PCT patent application
IL0 1/00992 or in unpublished copending PCT patent application IL04/00571 of the
Applicant filed 27 June 2004 (vide infra).
10 In an embodiment of the method of the present invention the precursor plate
comprises a multiwell plate and the picowell-bearing component is placed in a
respective well.
In an embodiment of the present invention, subsequent to the placing of the
picowells-bearing component on the precursor plate, walls of the plurality of wells are
15 attached to the precursor plate. Attaching includes the use of methods employing
adhesives or surface treatments such as plasma treatments.
In an embodiment of the present invention, subsequent to placing the picowells
on the precursor plate, the inside surface of picowells of the plurality of picowells are
coated with a layer of a coating material.
20 As noted above, whatever method is used for making a multiwell plate of the
present invention, it is often desired to coat the plurality of picowells, especially the
inside surface of picowells with some material. Coating the inside surface of a picowell
allows modification of the properties of the picowell, for example to reduce
cytotoxicity, to change physical properties such as solvent resistance or permeability or
25 to delay proliferation of cells held in a respective picowell. In an embodiment of the
method of the present invention, coating the inside surface of picowells comprises (i)
applying a precursor fluid to inside surfaces of the picowells; and (ii) solidifying the
precursor fluid so as to form the layer. Suitable methods of solidifying include but are
not limited to heating the precursor fluid, cooling the precursor fluid, polymerizing the
30 precursor fluid, cross-linking the precursor fluid, curing the precursor fluid, irradiating
the precursor fluid, illuminating the precursor fluid, gelling the precursor fluid, exposing
the precursor fluid to a fixative and waiting a period of time.
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In another embodiment of the method of the present invention, coating the inside
of the wells comprises (i) depositing a vapor of the coating material onto the inside
surface of the picowells thereby forming the layer of coating material.
In another embodiment of the present invention, coating the inside surface of the
wells comprises (i) depositing a vapor of a coating precursor material onto the inside
surface of the picowells; and (ii) solidifying the coating precursor material thereby
forming the layer of the coating material. Suitable methods of solidifying the coating
precursor material depend on the details of the specific embodiment and include but are
not limited to heating the coating precursor material, cooling the coating precursor
material, polymerizing the coating precursor material, cross-linking the coating
precursor material, curing the coating precursor material, irradiating the coating
precursor material, illuminating the coating precursor material, gelling the coating
precursor material, exposing the coating precursor material to a fixative and waiting a
period of time. In a preferred embodiment, the vapor of coating precursor material is a
vapor of para-xylylene molecules or derivatives thereof and the layer comprises the
polymerized para-xylylene molecules (or derivatives thereof). By para-xylylene
derivatives is meant a a molecule that is substantially a para-xylylene molecules having
any additional substituent on either or both aromatic rings.
According to the teachings of the present invention there is also provided a
device comprising an array of living cells held in a non-fluid matrix, where the matrix is
configured to maintain cell viability. Preferably, the living cells are physically held in
pockets in the matrix and there is substantially no bond between the living cells and the
matrix. In a preferred embodiment, the array is substantially planar having an upper
surface and a lower surface. In a preferred embodiment, one or both of the two surfaces
is transparent to at least one wavelength of light or range of wavelengths of light in the
ultraviolet, visible or infrared light spectrum.
In a preferred embodiment of the present invention, the matrix is configured to
substantially delay the proliferation of living cells held therein.
In an embodiment of the device of the present invention the matrix comprises a
material having an index of refraction similar to that of water. In a preferred
embodiment of the device present invention, the index of refraction of the matrix is less
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than about 1.4, less than about 1.38, less than about 1.36, less than about 1.35, less than
about 1 .34 or substantially equal to that of water.
One material from which a matrix is preferably made that generally has at least
some of the preferred properties described above is a gel, especially a hydrogel. Suitable
5 gels are as described above for gel picowells of a multiwell plate of the present
invention.
In a preferred embodiment of the present invention, the matrix further comprises
an active entity. A preferred active entity is an indicator, especially an indicator
configured to indicate a cell response to a stimulus, such as the release of a second
10 active entity.
According to the teachings of the present invention there is also provided a
method for handling living cells, comprising: (a) providing an ordered array of living
cells immobilized in a non-fluid matrix, the matrix configured to maintain cell viability;
(b) contacting the living cells with a stimulus; and (c) detecting a response of the cells to
15 the stimulus. The method of handling living cells of the present invention is preferably
implemented using the device of the present invention.
In an embodiment of the present invention, the matrix further comprises an
active entity. A preferred active entity is an indicator, especially an indicator configured
to indicate a cell response to a stimulus, such as the release of a second active entity.
20 In an embodiment of the present invention, part of the detecting a response
comprises contacting the matrix with an active entity. A preferred active entity is an
indicator, especially an indicator configured to indicate a cell response to a stimulus,
such as the release of a second active entity. In some embodiments, it is required to wait
a period of time so as to allow the contacted active entity to reach proximity with the
25 cells, for example by diffusion through the matrix.
In an embodiment of the present invention, detecting comprises detecting
emitted light, for example light emitted by a cell or from an indicator, for example by
fluorescence. In an embodiment of the present invention, detecting comprises detecting
light, for example light reflected, diffracted, passing through or passing by a cell or an
30 indicator.
According to the teachings of the present invention there is provided a method
for producing an ordered array of living cells in a non-fluid matrix, comprising: (a)
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PCT/IL2004/000661
providing a multiwell plate provided with a plurality of wells, the multiwell plate
including a plurality of picowells at the bottom of at least one well, the plurality of
picowells including picowells; (b) placing a suspension of a plurality of living cells in a
gellable fluid in the at least one well; (c) causing the living cells to settle into the
5 picowells so as to be held in respective picowells; and (d) gelling the gellable fluid so as
to make a gel cover, trapping the living cells between the picowells and the gel cover. In
an embodiment of the present invention, the picowells are made of a material
comprising a gel.
Generally, causing the living cells to settle into the picowells includes applying a
10 force to the cells, typical forces including gravitation, centrifugal forces, forces resulting
from the impact of photons on the cells {e.g., laser tweezers, application of a non-
focussed laser (see, for example, PALM, Microlaser Technologies AG, Bernried,
Germany)), or forces resulting from a pressure wave (such as produced by an ultrasonic
transponder).
15 In a preferred embodiment, prior to gelling the gellable fluid, it is ensured that
each picowell holds no more than one living cell. In another preferred embodiment,
prior to gelling the gellable fluid, it is ensured that each picowell holds no more than a
predetermined number of living cell or holds a predetermined number of living cells.
In a preferred embodiment, the gellable fluid is chosen so that upon gelling a
20 transparent gel is formed. In a preferred embodiment, the gellable fluid is chosen so that
upon gelling a hydrogel is formed.
Depending on the nature of the gellable fluid used, preferred methods of gelling
the gellable fluid include of heating the gellable fluid, cooling the gellable fluid,
irradiating the gellable fluid, illuminating the gellable fluid, contacting the gellable fluid
25 with a gelling reagent and waiting a period of time for the gellable fluid to gel. Gellable
fluids suitable for use in in implementing the method of the present invention include
but are not limited to agar gel solutions, agarose gel solutions, gelatin solutions, low
melting temperature agarose gel solutions, alginate gel solutions, room-temperature
Ca -induced alginate gel solutions and polysaccharide gel solutions. Depending on the
30 embodiment, a gellable fluid has a water content of greater than about 80% by weight,
greater than about 92% by weight, greater than about 95% by weight, greater than about
97% by weight and even greater than about 98% by weight. A preferred gellable fluid is
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an alginate solution where gelling the gellable fluid includes contacting the gellable
fluid with a gelling reagent, such as a gelling reagent including Ca 2+ ions. An additional
preferred gellable fluid is a low melting temperature agarose solution and gelling the
gellable fluid includes cooling the gellable fluid. In an embodiment of the present
5 invention, the gellable fluid further comprises an active entity. A preferred active entity
is an indicator, especially an indicator configured to indicate a cell response to a
stimulus, such as the release of a second active entity.
BRIEF DESCRIPTION OF THE DRAWINGS
10 The invention is herein described, by way of example only, with reference to the
accompanying drawings. With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present invention only, and
are presented in the cause of providing what is believed to be the most useful and
15 readily understood description of the principles and conceptual aspects of the invention.
In this regard, no attempt is made to show structural details of the invention in more
detail than is necessary for a fundamental understanding of the invention, the
description taken with the drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
20 In the drawings:
FIG. 1 (prior art) depicts a cell-chip device of PCT patent application
IL0 1/00992 including a transparent carrier;
FIG. 2 (prior art) is a reproduction of a photograph of a cell-chip device of PCT
patent application IL0 1/00992;
25 FIG. 3 (prior art) is a reproduction of a photograph of a cell-populated well-array
of a carrier of a cell-chip device of PCT patent application IL0 1/00992;
FIG. 4 (prior art) is a schematic depiction of a standard commerically available
96-well plate;
FIGS. 5A-5B are reproduction of photographs of a 96-well plate of the present
30 invention showing wells and picowells;
FIG. 6 is a reproduction of a scanning electron micrograph of an array of
picowells of a multiwell plate of the present invention;
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FIG. 7 is a schematic depiction of a side view of picowells of the present
invention configured as enclosures;
FIG. 8 is a reproduction of a scanning electron micrograph of the domes on a
nickel template used for the production of a plurality of picowells of the present
invention;
FIGS. 9A-9F are schematic depictions of steps of a method of the present
invention for making a multiwell plate of the present invention by contacting a template
bearing negatives of wells and a pluralities of picowells with a reversibly deformable
precursor material;
FIGS. 1 OA- 10C are schematic depictions of steps of a method of the present
invention for making a multiwell plate of the present invention by contacting a template
bearing negatives of a plurality of picowells with a reversibly deformable precursor
material inside a well of preexisting multiwell plate;
FIGS. 11A-11E are schematic depictions of steps of a method of the present
invention for making a multiwell plate of the present invention by contacting a template
bearing negatives of pluralities of picowells with a reversibly deformable precursor
material followed by attachment of a separate well-wall component;
FIGS. 12A-12D are schematic depictions of steps of a method of the present
invention for making a multiwell plate of the present invention by producing picowells
on a flat precursor plate using photolithography followed by attachment of a separate
well-wall component;
FIGS. 13A-13C are schematic depictions of steps of a method of the present
invention for making a multiwell plate of the present invention by producing pluralities
of picowells by photolithography inside wells of a preexisting multiwell plate;
FIGS. 14A-14C are schematic depictions of steps of a method of the present
invention for making a multiwell plate of the present invention by attaching preexisting
pico well-bearing carriers inside wells of a preexisting multiwell plate;
FIGS. 15A-15C are schematic depictions of steps of a method of the present
invention for making a multiwell plate of the present invention by attaching preexisting
picowell-bearing carriers to a substantially flat precursor plate followed by attachment
of a separate well-wall component;
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FIG. 16 is a schematic depiction of a device of the present invention being
substantially a 3 by 3 array of living cells held in a non- fluid matrix; and
FIG. 17 is a schematic depiction of a 96-well plate of the present invention
comprising arrays of living cells in a non- fluid matrix.
5
DETAILED DESCRIPTION OF THE INVENTION
The present invention is of a mulitwell plate having a plurality of picowells on
the bottom of the wells of the plate. The present invention is also of methods of
producing a mulitwell plate of the present invention. The present invention is also of a
10 device comprising an array of living cells held in a non-fluid matrix. The present
invention is also of a method of handling living cells by providing an ordered array of
living cells immobilized in a non-fluid matrix, contacting the living cells with a
stimulus; and detecting a response to the stimulus. The present invention is also of a
method of producing an ordered array of living cells.
15 The principles and uses of the teachings of the present invention may be better
understood with reference to the accompanying description, figures and examples. In
the figures, like reference numerals refer to like parts throughout.
Before explaining at least one embodiment of the invention in detail, it is to be
understood that the invention is not limited in its application to the details set forth
20 herein. The invention can be implemented with other embodiments and can be practiced
or carried out in various ways. It is also understood that the phraseology and
terminology employed herein is for descriptive purpose and should not be regarded as
limiting.
Generally, the nomenclature used herein and the laboratory procedures utilized
25 in the present invention include techniques from the fields of biology, chemistry and
engineering. Such techniques are thoroughly explained in the literature. Unless
otherwise defined, all technical and scientific terms used herein have the same meaning
as commonly understood by one of ordinary skill in the art to which the invention
belongs. In addition, the descriptions, materials, methods, and examples are illustrative
30 only and not intended to be limiting. Methods and materials similar or equivalent to
those described herein can be used in the practice or testing of the present invention. All
publications, patent applications, patents and other references mentioned are
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incorporated by reference in their entirety as if fully set forth herein. In case of conflict,
the specification herein, including definitions, will control.
As used herein, the terms "comprising" and "including" or grammatical variants
thereof are to be taken as specifying the stated features, integers, steps or components
5 but do not preclude the addition of one or more additional features, integers, steps,
components or groups thereof. This term encompasses the terms "consisting of and
"consisting essentially of.
The phrase "consisting essentially of or grammatical variants thereof when used
herein are to be taken as specifying the stated features, integers, steps or components but
10 do not preclude the addition of one or more additional features, integers, steps,
components or groups thereof but only if the additional features, integers, steps,
components or groups thereof do not materially alter the basic and novel characteristics
of the claimed composition, device or method.
The term "method" refers to manners, means, techniques and procedures for
15 accomplishing a given task including, but not limited to, those manners, means,
techniques and procedures either known to, or readily developed from known manners,
means, techniques and procedures by practitioners of the chemical, pharmacological,
biological, biochemical and medical arts. Implementation of the methods of the present
invention involves performing or completing selected tasks or steps manually,
20 automatically, or a combination thereof.
Herein, the term "active entity" is understood to include chemical, biological or
pharmaceutical entities including any natural or synthetic chemical or biological
substance that influences a cell with which the entity is in contact. Typical active
entities include but are not limited to active pharmaceutical ingredients, antibodies,
25 antigens, biological materials, chemical materials, chromatogenic compounds, drugs,
enzymes, fluorescent probes, immunogenes, indicators, ligands, nucleic acids, nutrients,
peptides, physiological media, proteins, receptors, selective toxins and toxins.
Herein, by "indicator" is meant any active entity that upon interaction with some
stimulus produces an observable effect. In the context of the present invention, by
30 stimulus is meant, for example, a specific second active entity (such as a molecule)
released by a cell and by observable effect is meant, for example, a visible effect, for
example a change in color or emission of light.
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Some embodiments of the present invention include components that are
transparent or are made of a transparent material. By "transparent" is meant that the
component or material is substantially transparent to radiation having a wavelength in at
least pat of the visible light spectrum, the ultraviolet light spectrum and/or of infrared
5 radiation, preferably the visible light spectrum.
It is important to note that some embodiments of the present invention are
related to embodiments of unpublished copending PCT patent application IL04/00571
of the Applicant filed 27 June 2004. In IL04/00571 are taught picowell-bearing carriers
having a variety of innovative features. One aspect of the teachings of PCT patent
10 application IL04/00571 is of picowells configured to influence cell proliferation of cells
held therein. In one embodiment, carriers having picowells of a changeable size is
taught. In another embodiment, carriers configured to delay proliferation of cells held
therein, for example by delaying or preventing cell adhesion, are taught. In another
embodiment, carriers configured so as to allow cells to grow into or through the carrier
1 5 are taught. The above-described embodiments are preferably implemented by making
the picowells of or coating the picowells with a material with the desired properties. In
some embodiments, the inner surface of a picowell with which a held cell makes contact
is configured to have the desired property, influence or effect. Preferred materials from
which to make carriers listed in PCT patent application IL04/00571 include
20 polydimethylsiloxane, elastomers (such as silicon rubber), polymerized para-xylylene
molecules, polymerized derivatives of para-xylylene molecules and gels (especially
hydrogels). In some embodiments, the inner surface of a picowell with which a held cell
makes contact is configured to have the desired property, influence or effect.
An additional aspect of PCT patent application IL04/00571 are the teachings of
25 a gel cover for picowell bearing components. The gel cover is configured to prevent
cells held in a picowell from exiting the picowell due to jostling, incidental fluid flows
or during movement of the carrier.
The advantages of a picowell-bearing carrier made of a gel, of a picowell gel-
cover or a gel carrier covered with a gel cover include, depending on the embodiment,
30 that active entities may be integrated into the gel, that active entities may be contacted
with the cell by diffusion through the gel, that diffusion of released compounds is
slowed down allowing identification of which cell released a given compound, that
10
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proliferation of cells held therein is delayed but once cells begin to proliferate, that
allows proliferation into and through the gel matrix.
As discussed hereinabove, a prior art multiwell plate is substantially a planar
device having an upper surface whereupon is found an array of wells configured to hold
a fluid containing cells or other entities. As noted above, multiwell plates generally have
a standard footprint of ca. 8.5 cm by ca. 12.5 cm. As noted above, the wells of a prior
art multiwell plate are generally distributed in a standard 2n by 3n rectangular packed
well-array, n being an integer. The standard multiwell plates have 6, 12, 48, 96, 384 or
even 1536 standard sized wells. The volume of the wells depends on the number of
wells and the depth thereof but is generally greater than 5 x 10"* liter (for a 1536 well
plate). In Figure 4 is depicted a top view of a prior art 96-well plate 42 from the top,
comprising 96 wells 44 arranged in a 8 by 12 array.
The present invention provides an improved multiwell plate where at the bottom
surface of at least one of the wells (preferably substantially all of the wells) are a
15 plurality of picowells. Figures 5 are top views of a multiwell plate of the present
invention. With no magnification, a plate of the present invention looks like prior art
plate 42 depicted in Figure 4. Magnification of a single well 44 of the 96 wells reveals
that at the bottom of a well 44 is found an array 18 of hexagonally packed 20 micron
hexagonal picowells 46, Figures 5A and 5B. In one embodiment, substantially the entire
bottom surface of such a well comprises picowells (as depicted in Figure 5A). In one
embodiment of the multiwell plate of the present invention, the picowell-containing
wells are homogenous, that is all have substantially the same size and arrangement of
picowells (as depicted in Figure 5A). In another embodiment of the multiwell plate of
the present invention, the picowell-containing wells are heterogenous, that is there is
variation between wells, for example variation in the size of the picowells, the
arrangement of the picowells or the material from which the picowells are made or with
which the picowells are coated.
The present invention also provides methods of making multiwell plates of the
present invention. According to one embodiment of the method of the present invention,
30 a preexisting multiwell plates is converted into a multiwell plates of the present
invention. According to another embodiment of the method of the present invention, a
multiwell plates of the present invention is fashioned in one piece, the wells and the
20
25
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picowells being integrally formed components of the multiwell plate. According to
another embodiment of the method of the present invention, a multiwell plates of the
present invention is fashioned by attaching a component or a plurality of components
that is substantially the walls defining the wells to a second component, where the
5 second component is substantially a plate bearing the picowells of the multiwell plate.
Multiwell plate of the present invention
As stated hereinabove, a multiwell plate of the present invention is substantially
a multiwell plate having a plurality of wells wherein at the bottom surface of at least one
10 well of the plate is found a plurality of picowells. Preferably, such a plate has a footprint
of a standard multiwell plate. Preferably, the wells of the plurality of wells of such a
plate are arranged in a manner similar or substantially identical to the arrangement of
wells of a standard multiwell plate, that is, a rectangular packing of 6n wells arranged in
a 2n by 3n array, where n is an integer greater than 0. Preferred are the most common
15 multiwell plate formats, that is, 6, 24, 96, 384 and 1536 wells, 96-wells and 384-wells
being most preferred. Preferably, the individual picowells of the plurality of picowells
are individually addressable. For ease of optical study and observation, it is preferred
that the bottoms of all the picowells of a certain well or of the entire plate be
substantially coplanar: coplanarity allows for optical observation of many cells (whether
20 by scanning or simultaneously using a wide-angle observation component) without the
need for time consuming and technically difficult to implement refocusing.
The use of a multiwell plate of the present invention allows efficient study of
pluralities of living cells as individuals.
On the one hand, standard accessories available in the art for manipulating and
25 using multiwell plates including robotic plate handlers, robotic fluid dispensers,
multipipettes, multifilters and the like are useable with the multiwell plates of the
present invention. Further, the format of the wells of prior art multiwell plates has
proven to be convenient for the performance of many simultaneous experiments in the
field of cellular biology, for example, during combinatorial studies.
30 On the other hand, cells placed in a well of a multiwell plate of the present
invention are held in the picowells of a respective plurality of picowells. The effect is
that a plurality of cells held in a multiwell plate of the present invention are arranged in
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a rationally ordered array. The rational arrangement of cells eases observation
(especially when the bottoms of the picowells are coplanar) and makes the cells more
easily observable as individuals (especially when the picowells are individually
addressable). Held cells are isolated from direct physical contact with other cells,
5 improving the quality of experimental results.
In an embodiment of the present invention, the picowells of the plurality of
picowells of a well are juxtaposed. By juxtaposed is meant that in an area where
picowells are found, most of the area is picowell area and little of the area is inter
picowell area. For example, in embodiments of the present invention, the inter picowell
10 area between two picowells is less than or equal to 0.35, 0.25, 0.15, 0.10 or even 0.06 of
the sum of the areas of the two picowells. In a preferred embodiment, the inter picowell
area is substantially nonexistent, for example when the rims of picowells are
substantially knife-edged. A picowell-array having substantially no inter picowell area
is seen in Figure 5B. In Figure 6, a reproduction of a scanning electron micrograph of a
15 picowell-array of a multiwell plate of the present invention having knife-edged rims is
shown. One advantage of juxtaposed picowells is that when cells are placed in a
respective well, the cells settle into picowells and do not settle onto inter picowell areas.
Further, when a plurality of juxtaposed picowells is used, a near-tissue density planar
array of cells is achieved. For example, an array of 10-micron wide hexagonal packed
20 knife-edged picowells has a picowell density of about 1.5 x 10 6 picowells cm" 1 .
Further, for reasons of a simple loading procedure and a high picowell density,
in a preferred embodiment of the present invention, a plurality of picowells covers
substantially the entire bottom surface of a respective well, as depicted in Figures 5.
As the teachings of the present invention are directed to cellular biology, it is
25 generally preferred that the picowells be small so as to avoid having a large number of
cells held in any one picowell. Thus, generally, the dimensions of the picowells are
generally less than about 200, 100, 50, 25 or even 10 microns. By dimensions is meant
the usual meaning of the word and is dependent on the shape of the picowell. For
example, for hexagonal or circular picowells, the term dimension refers to diameter. For
30 square or triangular picowells is meant the longest dimension of the square or triangle,
respectively. The exact dimensions of individual picowells depends on the type (and
consequently size) of cells to be studied and the types of experiments and studies that
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are to be performed. Since different types of cells have different sizes, generally a
multiwell plate of the present invention has picowells of a size to accommodate one or
more cells of the type to be studied. In some embodiments it is preferred that an
individual picowell be of a size so as to hold no more than one living cell of a certain
5 size. In other embodiments it is preferred that the picowell be of a size so as to held no
more than a predetermined number of cells of a certain size (e.g., two or three cells
simultaneously).
In some embodiments of the present invention, picowells are dimples or
depressions on the bottom surface of the inside of a well of a multiwell plate, as seen in
10 Figure 6. In other embodiments, depicted in side view in Figure 7, picowells 46 are
substantially enclosures of dimensions so that at least one cell 48 of a certain size is
containable, substantially entirely, within the enclosure, each enclosure having an
opening 50 at the surface, the opening defined by a first cross section of a size allowing
passage of cell of the certain size 48. The exact dimensions of the individual enclosures
15 depends on the type (and consequently size) of cells to be studied and the types of
experiments and studies that are to be performed. The volume of such enclosure
picowells is typically less than 1 x 10" 11 liter (corresponding to the volume of a 200
micron cube), less than 1 x 10* 12 liter (corresponding to the volume of a 100 micron
cube), less than 1 x 10" 13 liter (corresponding to the volume of a 50 micron cube), less
20 than 1 x 10" 14 liter (corresponding to the volume of a 25 micron cube) and even less than
1 x 10" 15 liter (corresponding to the volume of a 10 micron cube). The area of the first
cross section, corresponding to the size of the opening of a respective enclosure is
typically less than about 40000 micron 2 (corresponding to the area of a 200 micron
square), 10000 micron 2 (corresponding to the area of a 100 micron square), 2500
25 micron 2 (corresponding to the area of a 50 micron square), 625 micron 2 (corresponding
to the area of a 25 micron square) or even less than about 100 micron 2 (corresponding to
the area of a 10 micron square).
In embodiments of the present invention, all the picowells of all the pluralities of
picowells in all the wells of the multiwell plate of the present invention are substantially
30 identical in size. In embodiments of the present invention, the plurality of picowells in
one well is substantially different from the plurality of picowells in a second well. For
example, in an embodiment of the present invention the size of the picowells of the
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plurality of picowells in one well is different from the size of the picowells of the
plurality of picowells in a second well. In embodiments of the present invention, the
plurality of picowells in one well includes picowells of different sizes or shapes. For
example, in an embodiment of the present invention, one well includes 10 micron
5 picowells together with 20 micron micron picowells.
A multiwell plate of the present invention is made of any suitable material.
Suitable materials include but are not limited to ceramics, elastomers, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane, polyurethane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate, polystyrene,
10 polyvinyl chloride, rubber, silicon, silicon oxide and silicon rubber. In an embodiment
of the present invention, the bottom surface of the wells is made of any suitable
material. Suitable materials include but are not limited to ceramics, elastomers, epoxies,
glasses, glass-ceramics, metals, polymers, plastics, polycarbonates,
polydimethylsiloxane, polyethylenterephtalate glycol, polymethyl methacrylate,
15 polystyrene, polyurethane, polyvinyl chloride, rubber, silicon, silicon oxide and silicon
rubber.
In embodiments of the present invention, an entire plate of the present invention
and all components thereof are made of one material. In other embodiments, a plate of
the present invention is made up of a number of different materials, for example, as a
20 plurality of layers or as a coated structure.
In an embodiment of the present invention, the walls of wells are integrally
formed with the bottom surface of the wells. In embodiments, a multiwell plate of the
present invention comprises at least one distinct well-wall component attached to the
bottom surface. Such a distinct well-wall component is made of any suitable material.
25 Suitable materials include but are not limited to ceramics, elastomers, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polyurethane, polymethyl methacrylate,
polystyrene, polyvinyl chloride, rubber, silicon, silicon oxide and silicon rubber.
In embodiments of the present invention, a plurality of picowells is integrally
30 formed with the bottom surface of a respective well.
In embodiments of the present invention, a multiwell plate of the present
invention comprises at least one distinct picowell-bearing component bearing a plurality
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of picowells, the component resting in or attached to the bottom surface of a respective
well. A suitable distinct picowell-bearing component is a carrier comprising a plurality
of picowells disposed on a surface, such as a carrier described in PCT patent application
IL0 1/00992 or in unpublished copending PCT patent application IL04/00571 of the
5 Applicant filed 27 June 2004. Picowell-bearing components are made of any suitable
material, including reversibly deformable materials and irreversibly deformable
materials. Suitable materials include but are not limited to gels, hydrogels, waxes,
hydrocarbon waxes, crystalline waxes, paraffins, ceramics, elastomers, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane,
10 polyethylenterephtalate glycol, polymers, polymethyl methacrylate, polystyrene,
polyurethane, polyvinyl chloride, rubber, silicon, silicon oxide and silicon rubber.
In an embodiment of the multiwell plate of the present invention, a picowell-
bearing component comprises a gel, preferably a transparent gel, preferably a hydrogel.
Gel picowell-bearing components are discussed in detail in PCT patent application
15 IL04/00571. As will be discussed in detail hereinfurther, in general a gel picowell-
bearing component of the present invention is advantageously produced by placing a
gellable fluid in a well of an existing multiwell plate, contacting the gel with a template
including, amongst others, negatives of the picowells, and then gelled. Gels suitable for
use in making a picowell-bearing component of a plate of the present invention include
20 but are not limited to agar gels, agarose gels, gelatins, low melting temperature agarose
gels, alginate gels, room-temperature Ca 2+ -induced alginate gels and polysaccharide
gels. Depending on the embodiment, a suitable gel has a water content of greater than
about 80% by weight, greater than about 92% by weight, greater than about 95% by
weight, greater than about 97% by weight and even greater than about 98% by weight.
25 Two exceptionally preferred types of hydrogels are alginates and low melting
temperature agaroses.
Alginates are biologically compatible polysaccharide proteins that are fluid at
low calcium ion concentrations (e.g., [Ca 2+ ] < 1 fiM) but gel upon exposure to higher
concentrations of calcium ions (e.g., [Ca 2+ ] = 20 mM). An exceptionally suitable
30 alginate for implementing the teachings of the present invention is sodium alginate and
may be purchased, for example, from Pronova Biopoiymers (Drammen, Norway) as
Protanal LF120 1% in water or Protanal LF200 1% in water.
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Low melting temperature agaroses are biologically compatible gels that before
gelling are fluid at temperatures that do not harm living cells (e.g., 20°C), gel at low
temperatures that do not harm living cells (e.g., 4°C) and remain stable until well-above
temperatures used for studying living cells (40°C). An exceptionally suitable agarose for
5 implementing the teachings of the present invention that may be purchased, for
example, from Cambrex Bio Science Rockland Inc. (Rockland, ME, USA) is HGS-
LMP Agarose (catalogue nr. 50221).
In an embodiment, the gel includes an active entity. Suitable active entities
include, but are not limited to antibodies, antigens, biological materials, chemical
10 materials, chromatogenic compounds, drugs, enzymes, fluorescent probes,
immunogenes, indicators, ligands, nucleic acids, nutrients, peptides, physiological
media, proteins, receptors, selective toxins and toxins.
In an embodiment of the present invention, picowells have a bottom surface
made of a first material and borders, such as the borders delineating the picowells, made
15 of a second material, the second material being substantially different from the first
material. In an embodiment of the present invention the first material is substantially the
material from which the bottom of the well is made, for example when the bottom
surface of the picowell is substantially the bottom surface of the well. In an embodiment
of the present invention, the second material is a fixed photoresist material. As is
20 detailed hereinbelow, such a picowell structure is achieved by fixing a photoresist
material applied to a precursor plate. An advantage of such like plates is that featues
such as picowells having flat bottom surfaces are easily made.
In an embodiment of the multiwell plate of the present invention, picowells are
configured with an inside surface configured to delay proliferation of cells held therein,
25 for example by delaying adhesion of living cells thereto. Picowells configured to delay
proliferation of living cells held therein are discussed in detail in PCT patent application
IL04/00571. In an embodiment of the plate of the present invention, the inside of a
picowell comprises a material that delays adhesion of living cells thereto, that is the
picowell is substantially fashioned from the adhesion-delaying material or the inside of
30 the picowell is coated with the adhesion-delaying material. A suitable material to coat
the inside of a picowell or from which to make a picowell comprises
polydimethylsiloxane, is substantially polydimethylsiloxane or is substantially pure
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polydimethylsiloxane. Suitable polydimethylsiloxane resins for coating picowells or to
make picowells are commercially available and can be purchased, amongst others,
under the trade names RTV615 PDMS (GE Silicones, Wilton, CT, USA) and Sylgard
184 PDMS (Dow Corning Corporation, Midland, MI, USA).
5 In an embodiment of the multiwell plate of the present invention, bottom
surfaces pf the picowells comprise a material having an index of refraction similar to
that of water, that is an index of refraction of less than about 1.4, less than about 1,38,
less than about 1.36, less than about 1.35, less than about 1.34 or substantially equal to
that of water. Picowells having indicia of refraction similar to that of water are
10 discussed in detail in PCT patent application IL04/00571. An advantage of such
picowells is that observation of cells is simplified as the picowell walls are substantially
invisible and there is little, if any, scattering, reflection and diffraction of light, that
otherwise interferes with optical study of held cells, for example, during morphological
studies using a microscope.
15 In an embodiment of the present invention, the plurality of picowells comprises
picowells having an inner surface coated with a layer of a material. Suitable materials
for coating an inner surface of a picowell of a plate of the present invention include but
are not limited to gels, hydrogels, polydimethylsiloxane, elastomers, polymerized para-
xylylene molecules, polymerized derivatives of para-xylylene molecules, rubber and
20 silicon rubber. Picowells having coated inner surfaces are discussed in detail in PCT
patent application IL04/00571.
In an embodiment of the present invention, a plate of the present invention
further comprises a gel cover covering a plurality of the picowells, the cover made of a
gel. Suitable gels are as described herein. Gel picowell covers are discussed in detail in
25 PCT patent application IL04/0057 1 .
In an embodiment of the present invention, a multiwell plate of the present
invention further comprises at least one additional feature functionally associated with
the plurality of picowells, especially a microfluidic feature. Suitable microfluidic
features include but are not limited to channels, coupling elements, drains, fluid
30 channels, fluid reservoirs, input ports, membranes, microreactors, microvalves, output
ports, passages, plumbing routes, protruberances, pumps, transport channels and valves.
Other suitable features include but are not limited to light sources, magnetizable
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elements, optical components, optical fibers, optical filters, protuberances, fiducial
points and walls. Such an embodiment can be considered to be a multiwell plate of the
present invention that holds a carriers such as described in PCT patent application
IL0 1/00992 or in unpublished copending PCT patent application IL04/00571. Such an
5 embodiment allows performance of many and varied experiments to study living cells,
as described in the above references.
In an embodiment of the of the present invention, a multiwell plate further
comprises a cover slip, the cover slip and a plurality of picowells in a well configured so
as to allow the cover slip to rest above the plurality of picowells substantially in parallel
10 to the bottom surface of the respective well. Such a cover slip can include
microelectrodes to assist in manipulation of cells held in picowells, can be used in
conjunction with other features so as to provide a microfluidics system for the
picowells, or for other reasons as discussed in PCT patent application IL0 1/00992.
1 5 Methods of manufacture of a multiwell plate of the present invention
A multiwell plate of the present invention is produced using any suitable method
known in the art. Suitable methods include methods that employ one or more techniques
including but not limited to casting, embossing, etching, free-form manufacture,
injection-molding, microetching, micromachining, microplating, molding, spin coating,
20 lithography or photo-lithography.
The preferred methods of producing a multiwell plate of the present invention
are the methods of the present invention.
A first method of the present invention for making a multiwell plate of the
present invention is substantially by contacting a precursor material with a template, the
25 template having a negative of some of the features of the plate (especially the picowells)
thus creating the features in the precursor material. The features are subsequently fixed
in the precursor material making an incipient plate. After any further processing of the
incipient plate required (which may be limited to simply separating the template from
the incipient plate), the multiwell plate of the present invention is fashioned.
30 Depending on the precursor material, fixing includes, but is not limited to,
methods such as heating the precursor material, cooling the precursor material, curing
the precursor material, polymerizing the precursor material, cross-linking the precursor
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material, irradiating the precursor material, illuminating the precursor material, gelling
the precursor material, exposing the precursor material to a fixative and waiting a period
of time. By fixative is meant an agent that causes the precursor material to change to the
fixed state and is used herein as a general term for such materials as fixatives,
5 hardeners, polymerization / crosslinking / curing initiators, catalysts and agents. It is
important to note that in some cases a precursor material is produced by mixing two or
more components which thereafter change to a fixed state, for example, by simply
waiting a period of time.
In one preferred embodiment of the present invention, the precursor material is a
10 irreversibly deformable precursor material. Herein by irreversibly deformable precursor
material is meant a material that does not recover a shape after deformation and so there
is usually no need for a separate action to fix the features in the precursor material
beyond separating the produced multiwell plate from the template. In such cases, the
precursor material does not substantially chemically change subsequent to contact with
15 the template. Examples of suitable irreversibly deformable precursor materials include
waxes, paraffins, plastics, polymers and the like. In such an embodiment, a preferred
template is a stamp, and the contacting of the template with the precursor material is
substantially stamping the features of the multiwell plate into the precursor material,
preferably under controlled thermal conditions.
20 In another preferred embodiment of the present invention, the precursor material
is a reversibly deformable precursor material. Herein by reversibly deformable
precursor material is meant a material that is capable of recovering shape after
deformation and includes gellable fluids, polymerizable materials, powders, fluids and
thermoplastic materials.
25 In a preferred embodiment, the reversibly deformable precursor material is a
thermoplastic material at a pliable templerature. Subsequent to the contacting of the
template but before the contact is finished, the thermoplastic material is cooled, thus
fixing the desired features in the incipient multiwell plate.
In another preferred embodiment, the reversibly deformable precursor material
30 is a polymerizable material (e.g., a monomer solution, a crosslinkable polymer, a
vulcanizable polymers, a polymerizable fluids or a thermosetting resin). Subsequent to
the contacting of the template but before the contact is finished, the polymerizable
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material is polymerized, thus fixing the desired features in the incipient multiwell plate.
In such cases, the precursor material and the material from which the multiwell plate is
made are chemically dissimilar (for example, have the relationship of monomer to
polymer).
5 One preferred polymerizable precursor material is a non-cured
polydimethylsiloxane precursor mixture. A mixture of two polydimethylsiloxane
components (the prepolymer and curing agent) are mixed together in the desired ratio
(preferably about 10:1, but ratios between about 5:1 and about 20:1 are generally
suitable) to give a polydimethylsiloxane precursor mixture, the mixture degassed and
10 contacted with the template. The features are fixed by the curing of the mixture. Curing
of polydimethylsiloxane precursor generally takes place at room temperature for about
24 hours and, when desired, is accelerated by heating. For example it has been found
that multiwell plates of the present invention made of polydimethylsiloxane are ready
for further processing within 2 hours when cured at 60°C or within 15 minutes when
15 cured at 150°C. A detailed review of methods for the production of micronic features
such as picowells from polydimethylsiloxane suitable for implementing the teachings of
the present invention are known in the art and discussed, for example, in Ng et al. 9
Electrophoresis 2002, 23, 3461-3473 and Duffy et al., Anal. Chem. 1998, 70, 4974-
4984.
20 Another preferred polymerizable precursor material is urethane that is
polymerized to yield polyurethane.
Another preferred reversibly deformable precursor material is a gellable fluid.
After the gellable fluid is brought in contact with the template, the features are fixed by
gelling the gellable fluid to yield a gel. Most preferred are gellable fluids that produce a
25 hydrogel.
Gellable fluids known in the art include fluids that gel upon heating, fluids that
gel upon cooling, fluids that gel upon irradiation or illumination, fluids that gel as a
result of contact with a gelling reagent and fluids that gel after a period of time.
Preferred gellable fluids for implementing the teachings of the present invention include
30 solutions of agars, agaroses, gelatins, low melting temperature agaroses, alginates,
proteins, protein polysaccharides, Ca 2+ -inducable alginates (especially those that gel at
room temperature) and polysaccharides.
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i
One preferred gellable fluid is a low-melting temperature agarose solution. Such
a solution is fluid at temperatures that do not harm living cells {e.g., 20°C) and gel at
low temperatures that do not harm living cells {e.g., 4°C). An exceptionally suitable
agarose for implementing the teachings of the present invention that may be purchased,
5 for example, from Cambrex Bio Science Rockland Inc. (Rockland, ME, USA) is HGS-
LMP Agarose 0.5% in PBS (catalogue nr. 50221).
Another preferred gellable fluid is an alginate solution which gels upon contact
with a gelling reagent, the preferred gelling reagent being a solution having a Ca 2+ ion
concentration of greater than about 1 x 10" 6 M. An exceptionally useful gelling agent is
10 a 20 x 10" 3 M calcium gluconate solution. Suitable alginate solutions can be purchased
from Pronova Biopolymers (Drammen, Norway) , and include, for example, Protanal
LF 1 20 1 % in water and Protanal LF200 1 % in water.
The template having a negative of the features is, for example, a stamp or a
mold, and is generally made of any suitable material that is more rigid than a respective
15 precursor material. Suitable materials include but are not limited to reversibly
deformable materials, irreversibly deformable materials, ceramics, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate, paraffins,
polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon oxide, silicon rubbers
20 and wax.
The template is made, for example, using methods with which one skilled in the
art is acquainted such as casting, embossing, etching, free-form manufacture, injection-
molding, microetching, micromachining, microplating, molding, lithography or photo-
lithography.
25 In Figure 8, is shown a reproduction of a scanning electron micrograph of the
domes on a nickel stamp used as a template for the production of a multiwell plate of
the present invention. Seen is an array of hexagonally-packed domes that are the
negative of a hexagonal array of knife-edged picowells, such as seen in Figure 6. The
diameter of the domes at the intersection with the nickel surface is approximately 20
30 microns.
In some embodiments, other features created in the precursor material by the
contact of the template include features such as drains, channels, coupling elements,
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drains, fiducial points, fluid channels, fluid reservoirs, input ports, microreactors,
microvalves, passages, optical components, optical filters, output ports, plumbing
routes, protruberances, protruberances for supporting a cover slip, pumps, transport
channels, valves, walls and partial walls.
5 In some embodiments of the present invention, the wells of a multiwell plate of
the present invention are made by contacting a precursor material with a template. Steps
for producing a six-well plate of the present invention 52 according to a method of the
present invention are schematically depicted in Figures 9. In Figure 9A, a fluid and
therefore reversibly deformable precursor material 54 is provided. Precursor material 54
10 is a molten thermoplastic material. In Figure 9B, a template 56 substantially a nickel
stamp having features that are negatives of features of plate 52 including wells and
picowells is provided. In Figure 9C, template 56 is brought in contact with precursor
material 54 so as to form the features of plate 52 in precursor material 54. In Figure 9D,
the features of plate 52 are fixed in precursor material 54 by cooling so as to make
1 5 incipient plate 58. After sufficient cooling, template 56 is separated from incipient plate
58, Figure 9E. Incipient plate 58 undergoes whatever further processing is necessary to
ultimately yield plate 52 of the present invention, having pluralities of picowells 18 in
each one of six wells 44, Figure 9F.
In a preferred embodiment, a multiwell plate of the present invention is made by
20 making picowells (and other desired features) as described above inside the wells of a
preexisting multiwell plate. Suitable multiwell plates include but are not limited to
plates made of reversibly deformable materials, irreversibly deformable materials,
ceramics, epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates,
polydimethylsiloxane, polyethylenterephtalate glycol, polymers, polymethyl
25 methacrylate, polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon oxide and
silicon rubbers. In such a case, the precursor material is placed into each desired well of
the preexisting multiwell plate. A template is then placed inside the well so as to make
contact with the precursor material and the precursor material is fixed as described
above. Such an embodiment has the advantage that a commercially available multiwell
30 plate of any format (e.g., 6, 24, 96, 384 and 1536 wells) and of virtually any material
can be converted into a multiwell plate of the present invention. In such an embodiment
a template can be made and used for fixing picowells and other features in any number
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of wells including for each well separately or for all wells simultaneously. In this way, a
single multiwell plate of the present invention having different features (e.g., different
sized picowells) in different wells is easily made.
Steps for producing a six-well plate of the present invention 52 according to a
5 method of the present invention are schematically depicted in Figures 10. In Figure
10A, a fluid and therefore reversibly deformable precursor material 54 is placed in a
preexisting 6-well plate 60 having six wells 44. Precursor material 54 is a non-cured
polydimethylsiloxane precursor mixture (comprising a mixture of a prepolymer and a
curing agent). In Figure 10B, a template 56 substantially a nickel stamp having features
10 that are negatives of features of plate 52 found in wells 44 such as picowells and having
a size and shape to precisely fit in a well 44 is contacted with precursor material 54 in
each one of wells 44 sequentially so as to form the features of plate 52 in precursor
material 54 in each one of wells 44 sequentially. Template 56 is maintained in contact
with precursor material 54 in a given well 44 for so long as required for the desired
15 features to be fixed in precursor material 54 by solidification to be
polydimethylsiloxane. In Figure 10B it is seen that at the bottom surfaces of each one of
three wells 44a are found a plurality of nanowells 18 while at the bottom of each one of
three wells 44b is found non-fixed precursor material 54. Incipient plate 58 undergoes
whatever further processing is necessary to ultimately yield plate 52 of the present
20 invention, having pluralities of picowells 18 in each one of six wells 44, Figure 10C.
In another preferred embodiment, the template includes the negative of the
desired features such as picowells but not of the wells. The template is contacted with
the precursor material so as to form a substantially planar incipient plate having the
features the negatives of which are found on the template. Subsequently, a grid-like
25 component, being substantially the walls of the wells of the multiwell plate of the
present invention, is attached using an appropriate method, for example, adhesives (for
example, light curable adhesives, such as light curing adhesive 3051 or 3341
manufactured by Henkel Loctite Deutschland GmbH, Munchen, Germany) or surface
treatments such as anodic bonding, fusion bonding or plasma treatment such as plasma
30 discharge (exceptionally suitable for attaching polydimethylsiloxane, see Duffy et ai,
Anal Chem. 1998, 70, 4974-4984).
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Steps for producing a six-well plate of the present invention 52 according to a
method of the present invention are schematically depicted in Figures 1 1 . In Figure
11 A, a fluid and therefore reversibly deformable precursor material 54 is provided.
Precursor material 54 is a non-cured polydimethylsiloxane precursor mixture
5 (comprising a mixture of a prepolymer and a curing agent). In Figure 1 1 A, a template
56 substantially a nickel stamp having features that are negatives of features of plate 52
such as picowells, but not wells is also provided. In Figure 1 IB, template 56 is brought
in contact with precursor material 54 so as to form the features of plate 52 in precursor
material 54. Template 56 is maintained in contact with precursor material 54 for so long
10 as required for the desired features to be fixed in precursor material 54 by solidification
to be polydimethylsiloxane and thus to produce a substantially planar incipient plate 58
having, amongst other features, six pluralities of nanowells 18, Figure 11C. In Figure
1 ID, a grid-like component 64, being substantially the walls of wells 44 of plate 52 of
the present invention is provided. Attachment of grid-like component 64 to substantially
15 planar incipient plate 62 using adhesive (e.g., light curing adhesive 3051 manufactured
by Henkel Loctite Deutschland GmbH, Munchen, Germany) and whatever further
processing is necessary ultimately yields plate 52 of the present invention, having
pluralities of picowells 18 in each one of six wells 44, Figure 1 IE.
Another preferred method of making a multiwell plate of the present invention
20 includes photolithography of a photoresist material placed on a substrate, a
commercially available process (for example, from Micro Resist Technology GmbH,
Berlin, Germany) with which one skilled in the art is well-acquainted.
In brief, a high aspect ratio photoresist material (e.g., SU-8 thick photoresist
fluid, MicroChem Corporation, Newton MA, USA) is placed on a precursor plate as a
25 uniformly thick film. A preferred method of achieving a uniformly thin film of a
photoresist fluid on a precursor plate is by spin coating, that is, the photoresist fluid is
placed on a surface of the precursor plate and the precursor plate is rotated about an axis
that is perpendicular to the surface of the substrate on which the photoresist fluid was
placed. As a result of the rotation the photoresist fluid forms a uniformly thick film on
30 the precursor plate, typically between about 5 microns and about 20 microns thick.
Once a film of uniform thickness of photoresist material is achieved, the photoresist
material is illuminated through a mask, the mask being substantially a template or
f
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master of the features which are desired to be fixed in the photoresist material including
the desired picowells. Developing of the precursor with the selectively fixed film
removes the non-fixed areas of the film. In such a way features of a multiwell plate of
the present invention are made up of a fixed photoresist layer resting on a precursor
5 plate where the features of the multiwell plate are carved into the photoresist layer and
the bottom of the features (such as picowells) is the surface of the precursor plate. Using
a photolithography method, picowells and other features are easily produced, including
features having a flat-bottom surface.
It is important to note that in addition to picowells, any suitable feature known in
1 0 the art and discussed herein for multiwell plates of the present invention or for picowell-
bearing carriers described in PCT patent application IL 01/00992 or PCT patent
application IL 04/00571 can also be added using the photoresist method. Such features
include but are not limited to channels, coupling elements, drains, fluid channels, fluid
reservoirs, input ports, microreactors, microvalves, passages, output ports, plumbing
15 routes, protruberances, pumps, transport channels, valves, walls and fiducial points.
The material from which the precursor plate is made can be any suitable
material. Suitable materials include but are not limited to ceramics, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane, polymers,
polyethylenterephtalate glycol, polymethyl methacrylate, polystyrene, polyurethanes,
20 polyvinyl chloride, silicon and silicon oxide.
Steps for producing a six-well plate of the present invention 52 according to a
method of the present invention are schematically depicted in Figures 12. In Figure
12 A, a flat precursor plate 66 is provided. In Figure 12B, the upper surface of flat
precursor plate 66 is coated with a uniformly thin film of a precursor material 54,
25 precursor material 54 being a photoresist fluid (e.g., SU-8 thick photoresist fluid,
MicroChem Corporation, Newton MA, USA). In Figure 12C, precursor material 54 is
illuminated through mask 68 using light source 70 so that features such as picowells are
fixed in precursor material 54. After the features are fixed, incipient plate 58 is
developed so as to remove non fixed photoresist material and undergoes any further
30 processing necessary including attachment of a grid-like component 64 to ultimately
yield plate 52 of the present invention, having pluralities of picowells 18 in each one of
six wells 44, Figure 12D. '
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In a preferred embodiment, the precursor plate comprises a multiwell plate. In
such a case, the photoresist material (preferably a photoresist fluid) is placed into each
desired well of the precursor plate and the photoresist material fixed by illumination as
described above. Such an embodiment has the advantage that a commercially available
5 multiwell plate of any format (e.g., 6, 24, 96, 384 and 1536 wells) and of virtually any
material can be converted into a multiwell plate of the present invention. In such an
embodiment a mask can be made and used for fixing picowells and other features in any
number of wells including for each well separately or for all wells simultaneously. In
this way, a single multiwell plate of the present invention having different features (e.g.,
10 different sized picowells) in different wells is easily made.
Steps for producing a six-well plate of the present invention 52 according to a
method of the present invention are schematically depicted in Figures 13. In Figure
13 A, a precursor material 54 is placed in an existing six-well plate 60 having six wells
44. Precursor material 54 is a photoresist fluid (e.g., SU-8 thick photoresist fluid,
15 MicroChem Corporation, Newton MA, USA). In Figure 13B, a probe 72 tipped with a
mask 74 and provided with a light source 70 having a size and shape to precisely fit in a
well 44 is brought in proximity with precursor material 54 in each one of wells 44
sequentially. During the time that mask 74 is in proximity with precursor material 54,
light source 70 is activated so that features such as picowells are fixed in precursor
20 material 54 in a respective well 44. In Figure 13B it is seen that at the bottom surfaces
of each one of three wells 44a are found a fixed plurality of nanowells 18 while at the
bottom of each one of three wells 44b is found non-fixed precursor material 54. After
features are fixed in all desired wells 44, incipient plate 58 is developed so as to remove
non fixed photoresist material and undergoes any further processing necessary.
25 Ultimately a plate 52 of the present invention is formed having pluralities of picowells
18 in each one of six wells 44, Figure 13C.
A preferred method for producing a multiwell plate of the present invention is
by producing a substantially planar incipient plate where substantially the entire upper
surface is provided with an array of picowells, whether by contact with a temple, by
30 photoresist or other methods. Subsequently a well-wall component or plurality of
components is attached to the upper surface. The well-wall component thereby defines
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the plurality of wells and the upper surface of the precursor plate is substantially the
bottom surface of the wells whereupon the picowells are found.
Another preferred method of making a multiwell plate of the present invention
comprises attaching one or more picowell-bearing components to a precursor plate
5 using an appropriate method, for example, using an adhesive or a surface treatment such
as a plasma treatment, for example as described above. A preferred picowell-bearing
component is a carrier comprising a plurality of picowells disposed on a surface.
Preferred carriers include those described in PCT patent application IL 01/00992 or
PCT patent application IL 04/00571.
10 In a preferred embodiment, the precursor plate comprises a multiwell plate. In
such a case, one or more picowell-bearing components are placed into one or more
wells of the precursor plate and attached using a suitable method. Such an embodiment
has the advantage that a commercially available multiwell plate of any format (e.g., 6,
24, 96, 384 and 1536 wells) and of virtually any material can be converted into a
15 multiwell plate of the present invention. In such a way many picowell-bearing
components of different types are prefabricated, for example by mass production and
placed as desired in as many wells of the precursor plate as desired. In this way, a single
multiwell plate of the present invention having different features {e.g., different sized
picowells) in different wells is easily made.
20 Steps for producing a six-well plate of the present invention 52 according to a
method of the present invention are schematically depicted in Figures 14. In Figure
14 A, an adhesive 76 (e.g., light curing adhesive 3051 manufactured by Henkel Loctite
Deutschland GmbH, Munchen, Germany) is placed in wells 44 of an existing six-well
plate 60 having six wells 44. In Figure 14B, carriers 26, such as carrier 26 depicted in
25 Figure 2, are placed in each one of six wells 44 and illuminated with light source 70.
Adhesive 76 is cured by exposure to light produced by light source 70, attaching each
one of six carriers 26 inside a respective well 44, producing a plate 52 of the present
invention, having pluralities of picowells 18 in each one of six wells 44, Figure 14C.
In another preferred embodiment, the precursor plate comprises a substantially
30 planar plate, preferably having the same footprint of a multiwell plate (ca. 8.5 cm by ca.
12.5 cm). The picowell-bearing components are placed in appropriate locations on the
precursor plate corresponding to the locations of one or more wells of the ultimately
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made multiwell plate and attached using a suitable method. Subsequently, a grid-like
component, being substantially the walls of the wells of the multiwell plate of the
present invention, is attached using an appropriate method, for example, using an
adhesive or a surface treatment such as a plasma treatment.
5 Steps for producing a six-well plate of the present invention 52 according to a
method of the present invention are schematically depicted in Figures 15. In Figure
1 5 A, a flat precursor plate 66 is provided. In Figure 1 5B, carriers 26, such as carrier 26
depicted in Figure 2, are attached to flat precursor plate 66, for example using an
adhesive. Attachment of grid-like component 64 to incipient plate 58 and whatever
10 further processing is necessary ultimately yields plate 52 of the present invention,
having pluralities of picowells 18 in each one of six wells 44, Figure 15C.
It is important to note that in embodiments of the method of making a multiwell
plate of the present invention by placing (and optionally attaching) preformed picowell-
bearing components to a precursor plate, it is often advantageous that a given picowell-
15 bearing component have dimensions similar or substantially identical to that of a well in
which the picowell-bearing component is attached. Such dimensions allow more exact
placement of the picowell-bearing component in the well.
Some embodiments of the multiwell plate of the present invention comprise
picowells where the inside surface of the picowells (with which held cells potentially
20 make physical contact) is coated with a layer of some desired coating material, for
example a coating material that influences the proliferation of living cells as described
in PCT patent application IL04/00571 .
One skilled in the art is acquainted with many ways and many coating materials
with which to coat an inside surfaces of picowells of a multiwell plate of the present
25 invention.
One preferred method of coating inside surfaces of picowells of a multiwell
plate of the present invention, applicable to virtually any multiwell plate produced by
virtually any method, comprises contacting a precursor fluid with the inside surface of
the picowells and subsequently solidifying the precursor fluid, forming the layer of the
30 coating material. Depending on the nature of (he precursor fluid, solidifying is
performed by any number of methods including but not limited to heating the precursor
fluid, cooling the precursor fluid, polymerizing the precursor fluid, cross-linking the
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precursor fluid, curing the precursor fluid, irradiating the precursor fluid, illuminating
the precursor fluid, gelling the precursor fluid, exposing the precursor fluid to a fixative
or waiting a period of time.
One preferred method of coating the inside surfaces of picowells of a multiwell
5 plate of the present invention, applicable to virtually any multiwell plate produced by
virtually any method, is by vapor deposition. Vapor deposition involves the deposition
of materials such as molecules or atoms onto a surface at low pressures and is
characterized by the production of evenly thin coatings on a surface, such as the inner
surface of a picowell of a multiwell plate of the present invention.
10 In one embodiment of vapor deposition to the inside surfaces of picowells of a
multiwell plate of the present invention, the atoms or molecules that make up the
coating material are deposited. In another embodiment of vapor deposition, the atoms or
molecules that comprise a precursor of the coating material are deposited on the inside
surfaces of the picowells, followed by solidifying the coating precursor material thereby
15 forming the layer of coating material. Solidifying of the coating precursor material to
form the layer of coating material is performed by any number of methods including but
not limited to heating the coating precursor material, cooling the coating precursor
material, polymerizing the coating precursor material, cross-linking the coating
precursor material, curing the coating precursor material, irradiating the coating
20 precursor material, illuminating the coating precursor material, gelling the coating
precursor material, exposing the coating precursor material to a fixative and waiting a
period of time.
A preferred coating material for coating the inside surfaces of picowells of a
multiwell plate of the present invention is made of polymerized para-xylylene
25 molecules (or derivatives thereof, specifically where one or more hydrogens, especially
aromatic hydrogens of either or both aromatic rings are substituted) deposited by vapor
deposition, a coating commercially known as Parylene® (available for example from
V&P Scientific, Inc., San Diego, CA, USA). Parylene® is preferred not only for cell
proliferation influencing properties but also for the fact that Parylene® coatings are
30 bacteria resistant, fungus resistant, transparent, have a low permeability, acid and base
resistant, uniform, thin (typically 0.1-1 micron) and without voids even when a coated
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surface includes configurations with sharp edges, points, flat surfaces, crevices or
exposed internal surfaces.
The teachings of the present invention provide the possibility of providing a
5 device useful in the field of cellular biology. A device of the present invention
comprises an array of living cells held in a non-fluid matrix, the matrix configured to
maintain cell viability. Generally, the matrix of a device of the present is configured to
maintain cell viability. To maintain cell viability, a matrix is generally non-cytotoxic
and allows transport of molecules necessary for cell survival and metabolism, such as
10 nutrients, gases, ions and waste to and from a living cell held therein. A suitable matrix
is a matrix comprising a gel, preferably a hydrogel. Preferably, the living cells are
physically held in pockets in the matrix, especially free-floating in a physiological fluid
in the pockets. To reduce any influence on the reactions of the cells, there is preferably
substantially no bond (e.g., chemical bond) between the living cells and the matrix.
15 In Figure 16 is depicted a device of the present invention 72, being substantially
nine living cells 74 floating inside pockets 76 inside a hydrogel matrix 78.
In a preferred embodiment, to simplify use of the device, the array is
substantially planar having an upper surface and a lower surface. In a preferred
embodiment of the present invention, by substantially planar is meant that substantially
20 all living cells of the array are arranged in a single unique plane. In another preferred
embodiment of the present invention, by substantially planar is meant that substantially
all the living cells are arranged in two or more planes.
To ease observation of the cells or detection of signals associated with the cells,
in a preferred embodiment of the device of the present invention, one or both of the two
25 surfaces of the device is transparent to at least one wavelength of light, including a
wavelength of light of the ultraviolet spectrum, the visible spectrum or the infrared
spectrum. Further, to ease observation of the cells or detection of signals associated with
the cells, in a preferred embodiment of the present invention, the matrix comprises a
material having an index of refraction substantially similar to that of water. By
30 substantially similar is meant an index of refraction of less than about 1 .4, less than
about 1.38, less than about 1.36, less than about 1.35 or even less than about 1.34.
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In a preferred embodiment of the present invention, the matrix is configured to
substantially delay proliferation of living cells held therein. Configuration of a matrix so
as to substantially delay proliferation of the living cells is taught in PCT IL04/00571. A
preferred method of configuring a matrix to substantially delay proliferation is to make
5 at least part of the matrix from a material that has proliferation delaying properties. A
preferred such material is a gel, especially a hydrogel.
In a preferred embodiment of the present invention, the matrix comprises an
active entity, especially an indicator. By indicator is meant an active entity configured
to indicate a cell response to a stimulus, for example a molecule that is chromatogenic
10 or fluorogenic upon exposure to some compound released by a given living cell held in
the device upon exposure to some stimulus.
The unique characteristics of a device of the present invention are better
understood by comparing the device to living tissue on the one hand and prior art arrays
of living cells on the other hand.
15 As is known to one skilled in the art, living tissue can be considered to comprise
living cells held within a matrix. Further living tissue can be maintained living for an
extended period of time. It is therefore known in the art to use devices incorporating
living tissues in a device for assaying cell response to stimuli. However, in living tissue
the cells are not in an array; the cells are not distinct from each other making
20 identification of individual cell response difficult if not impossible especially under high
throughput conditions; cells are not individually addressable so a cell of interest must be
maintained under continuous observation; cells are not coplanar making visual study
time-consuming due to the need for refocusing; cells are in contact with each other so
that one cell may influence other neighboring cells; living tissue cannot be engineered
25 as desired to hold specific different cells in a desired spatial relationship to each other;
cells in living tissue proliferate, meaning that the properties of a device including living
tissue are not well-defined and change over time. Depending on the embodiment, a
device of the present invention provides overcomes some or all of these disadvantages.
As discussed in the introduction, prior art arrays of cells all have a number of
30 critical disadvantages. In some prior art arrays, the cells are bound to some object,
whether by native adhesion or by some non-native chemical bond or attraction. Binding
a cell necessarily compromises the response of cell to stimuli. In other prior art cell
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arrays, there is nothing keeping cells from moving from a designated location so that
there is no way to ensure that array integrity is maintained. This is exceptionally
significant when cell apoptosis or other death processes occurs or when the cell array is
moved. The device of the present invention provides, in contrast to prior art devices, a
5 cell array that is robust during any cell process including cell death and during
movement of the device itself.
The device of the present invention is exceptionally useful for implementing
certain manipulations of living cells. For example, a device of the present invention,
being substantially an ordered array of selected cells, is made in a first location such as
10 a laboratory,. The device is transported to a remote location, for example to a second
laboratory, in a space vehicle or to the location of a suspected environmental disaster. A
sample is contacted with the array of living cells of the device (for example, by
diffusion through the matrix). The reaction of the cells is observed indicating something
about the nature of the sample or of the living cells. As is clear to one skilled in the art,
1 5 the device of the present invention is useful for transporting ordered cell arrays. As is
clear to one skilled in the art, the device of the present invention is also useful as an
indicator or assay device.
The method of the present invention comprises providing an ordered array of
living cells immobilized in a matrix, the matrix configured to maintain cell viability; (b)
20 contacting the living cells with a stimulus; and (c) detecting the response of the living
cells to the stimulus.
To simplify detection or observation of the response, in a preferred embodiment
of the present invention, the matrix comprises an active entity, especially an indicator,
as described above. In another preferred embodiment (alone or together with an active
25 entity of the matrix), the matrix is contacted with an active entity, preferably an active
entity in solution. Generally, subsequent to contacting an active entity with the matrix, it
is necessary to wait a period of time in order to allow the active entity to reach the
proximity of the cells, for example by diffusion.
Although there are many methods for detecting the response of the living cells to
30 a stimulus, the preferred method involves the detection of light. By detection of light is
meant detection of emitted light (for example, light emitted by an indicator or light
emitted by a given cell). By detection of light is also meant detection of light that has
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interacted with a given cell, the vicinity of a given cell, or an indicator in the vicinity of
a given cell where the light is indicative of the cell response. Clearly such detecting
includes detection of fluorescence, differential polarization and optical inspection of a
cell.
5 A device of the present invention is a preferred device for implementing the
method of the present invention. That said, the present invention also provides a general
method for producing an ordered array of living cells useful, for example, in
implementing the method of the present invention using a multiwell plate of the present
invention. The method of producing an ordered array of living cells, comprises: (a)
10 providing a multiwell plate of the present invention, (b) placing a suspension of a
plurality of living cells in a gellable fluid in at least one well provided with picowells;
(c) causing the living cells to settle into the picowells so as to be held in respective
picowells; and (d) gelling the gellable fluid so as to make a gel cover, trapping the
living cells between the picowells and the gel cover. In a preferred embodiment, the
15 picowells are made of a material comprising a gel, preferably a hydrogel.
Generally, causing the cells to settle into the wells includes applying a force to
the cells, typical forces including gravitation, centrifugal forces, forces resulting from
the impact of photons on the cells (e.g., laser tweezers, application of a non-focussed
laser (see, for example, P.A.L.M. Microlaser Technologies AG, Bernried, Germany)),
20 or forces resulting from a pressure wave (such as produced by an ultrasonic
transponder). Most preferred is the application of centrifugal force, vide infra.
As stated above, once the cells have settled in a respective picowell, it is
preferred to gel the gellable fluid so as to form a cover on the picowells. As a result, the
cells are held snugly, without excessive physical stress, between the inside of a
25 respective picowell and the surrounding gel cover. An appropriate method of gelling a
gellable fluid is dependent on the nature of the gellable fluid and includes methods such
as heating the gellable fluid, cooling the gellable fluid, irradiating the gellable fluid,
illuminating the gellable fluid, contacting the gellable fluid with a gelling reagent and
waiting a period of time for the gellable fluid to gel.
30 It is generally preferred to usea gellable fluid that forms a hydrogel upon gelling.
Exceptionally suitable gellable fluids are fluids that comprise a material selected from
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the group consisting of agars, agaroses, gelatins, low melting temperature agaroses,
alginates, room-temperature Ca 2+ -inducable alginates and polysaccharides.
It is preferred that a gellable fluid that gels under conditions that are conducive
for cell survival be used for implementing the teachings of the present invention. One
5 preferred gellable fluid is an alginate solution. Alginates are biologically compatible
polysaccharide proteins that are fluid at low calcium ion concentrations (e.g., [Ca 2+ ] < 1
fiM) but gel upon exposure to higher concentrations of calcium ions (e.g., [Ca 2+ ] = 20
mM). An exceptionally suitable alginate for implementing the teachings of the present
invention is sodium alginate and may be purchased, for example, from Pronova
10 Biopolymers (Drammen, Norway) as Protanal LF120 1% in water or Protanal LF200
1% in water.
Another preferred gellable solution is a solution of low melting temperature
agarose. Low melting temperature agaroses are biologically compatible gels that before
gelling are fluid at temperatures that do not harm living cells (e.g., 20°C), gel at low
15 temperatures that do not harm living cells (e.g., 4°C) and remain stable until well-above
temperatures used for studying living cells (40°C). An exceptionally suitable agarose for
implementing the teachings of the present invention that may be purchased, for
example, from Cambrex Bio Science Rockland Inc. (Rockland, ME, USA) is HGS-
LMP Agarose (catalogue nr. 50221).
20 It is important to note that for maximal utility of a produced device, it is
desirable to ensure that substantially each pocket holds no more than one living cell or
no more than a predetermined number of cells. This is most easily achieved by ensuring
that the picowells of the picowell array are juxtaposed and that a given picowell is of a
size to accommodate no more than one living cell (or the predetermined number of
25 cells). Generally when a suspension of cells with a number of cells greater than the
number of picowells is placed in proximity of juxtaposed picowells and the cells
allowed to settle, all picowells will be filled but there will be excess cells "stacked" on
top of cells held in picowells. A preferred method for preventing such "stacking" is that
the suspension brought in proximity of the picowells has approximately a predetermined
30 number of cells. It has been found that when the number of cells in the suspension is
approximately equal to the number of picowells (or the product of the number of
picowells and the number of cells desired to be held in each picowell), there is
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substantially the desired number of cells per picowell, with only minimal stacking of
cells on top of already fully occupied picowells.
In a preferred embodiment of the method of the present invention, the gellable
fluid includes an active entity, especially an indicator, as described above. In such a
5 way, the active entity becomes an integral part of the matrix of a device produced
according to the method of the present invention.
In some embodiments of the method of the present invention, some or all of the
wells of a multiwell plate are provided with picowells. In some embodiments, all of the
picowells of the multiwell plate are substantially the same. In some embodiments, all of
10 the picowells in one well are substantially the same, but are different from picowells in
other wells. In some embodiments, in a given well, there are found two different types
of picowells. By different is meant, for example, have a different size or comprise
different active ingredients.
In a specific example, a 96-well glass plate 52 of the present invention is
15 provided, Figure 17. In plate 52, all wells 44 are provided with a plurality of integrally
formed hexagonally-packed knife-edged hexagonal picowells. The picowells in each
one of wells 44 of rows A, B, C and D have a diameter of 10 micron while the plurality
of picowells 18 in each one of wells 44 of rows in rows E, F, G and H have a diameter
of 20 micron.
20 Using an automatic liquid dispensing robot (Automated Microplate Pipetting
Systems - Precision™ XS Microplate Sample Processor, Bio-Tek Instruments,
Vinooski, VT, USA) different suspensions of living cells in liquid solutions of a
gellable solution, comprising a low melting temperature agarose (e.g., HGS-LMP
Agarose of Cambrex Bio Science Rockland Inc., Rockland, ME, USA) are added to the ;
25 wells of each row. The cells suspended in the solution dispensed in rows A, B, C and D
are peripheral lymphocytes having a diameter of about 5 to 7 microns. The cells
suspended in the solution dispensed in rows E, F, G and H are Jurkat T cell line cells
having a diameter of about 15-20. The number of cells dispensed in each well 44 is
about 95% of the number of picowells in that well. In addition, the solutions dispensed
30 into a given well 44 also include active reagents:
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a. in the solutions dispensed in rows A, B, E and F a first indicator for measuring
mitochondrial membrane potential is dispensed (lOOnM tetramethyl rhodamine methyl
ester);
b. in the solutions dispense in rows C, D, G and H a second indicator for
5 measuring intracellular levels of reactive oxygen species is dispensed (10 jxM
dichlorodihydro fluorescein diacetate);
Once all cell suspensions are dispensed, 96-well plate 52 is transferred to a
centrifuge provided with a cooling unit and centrifuged so as to cause dispensed cells to
settle into picowells. After sufficient time for cell settling, centrifugation is stopped and
10 the cooling unit is activated so as to bring the temperature of the gel to about 4°C and
thus initiate gelling. When sufficient time has passed for complete gelling of the
gellable fluid, the multiwell plate is used for examining metabolism during cell death.
To rows B, D, F and H a third active agent (50 solution of hydrogen peroxide as
apoptosis inducer) is added to each one of wells 44 of rows B, D, F and H, During the
15 time it takes for the third active agent to diffuse through the gel cover, plate 52 is
transferred to an observation unit configured to detect the intensity of color developed
in each picowell. Comparision of the development of color in wells of row B (with A as
control) shows the development of mitochondrial membrane potential as a result of
apoptosis of peripheral lymphocytes. Comparision of the development of color in wells
20 of row C (with D as control) shows the development of intracellular levels of reactive
oxygen species as a result of apoptosis of peripheral lymphocytes. Comparision of the
development of color in wells of row F (with E as control) shows the development of
mitochondrial membrane potential as a result of apoptosis of Jurkat T cells.
Comparision of the development of color in wells of row H (with G as control) shows
25 the development of intracellular levels of reactive oxygen species as a result of
apoptosis of Jurkat T cells.
Since each of the cells is held in a respective picowell, no cell is lost during the
apoptosis process. Since the cells are held in a substantially planar array of cells, the
exact number of cells and distribution of reactions is accurately monitored.
30
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It is appreciated that certain features of the invention, which are, for clarity,
described in the context of separate embodiments, may also be provided in combination
in a single embodiment. Conversely, various features of the invention, which are, for
brevity, described in the context of a single embodiment, may also be provided
separately or in any suitable subcombination. Although the invention has been described
in conjunction with specific embodiments thereof, it is evident that many alternatives,
modifications and variations will be apparent to those skilled in the art. Accordingly, the
present invention is intended to embrace all such alternatives, modifications and
variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification
are herein incorporated in their entirety by reference into the specification, to the same
extent as if each individual publication, patent or patent application was specifically and
individually indicated to be incorporated herein by reference. In addition, citation or
identification of any reference in this application shall not be construed as an admission
that such reference is available as prior art to the present invention.
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WHAT IS CLAIMED IS
1. A multiwell plate comprising a plurality of wells wherein at the bottom
surface of at least one well of said plurality of wells is a plurality of picowells.
2. The plate of claim 1 , having a footprint of a standard multiwell plate.
3. The plate of claim 1, wherein said plurality of wells comprises 6n wells
arranged in a 2n x 3n array, where n is an integer greater than 0.
4. The plate of claim 1, wherein wells of said plurality of wells are
rectangularly packed.
5. The plate of claim 3, wherein said plurality of wells is selected from the
group consisting of 6, 24, 96, 384 andT536 wells.
6. The plate of claim 3, wherein said plurality of wells is 96 wells.
7. The plate of claim 3, wherein said plurality of wells is 384 wells.
8. The plate of claim 1, wherein said plurality of picowells comprises
individually addressable picowells.
9. The plate of claim 1, wherein the bottoms of all picowells in a said well
of the plate are substantially coplanar.
1 0. The plate of claim 1 , wherein the bottoms of all picowells of the plate are
substantially coplanar.
1 1 . The plate of claim 1 , wherein picowells of said plurality of picowells are
juxtaposed.
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12. The plate of claim 11, wherein the interwell area between two said
picowells is less then about 0.35 the sum of the areas of said two picowells.
13. The plate of claim 11, wherein the interwell area between two said
picowells is less then about 0.25 the sum of the areas of said two picowells.
14. The plate of claim 11, wherein the interwell area between two said
picowells is less then about 0.15 the sum of the areas of said two picowells.
15. The plate of claim 11, wherein the interwell area between two said
picowells is less then about 0.10 the sum of the areas of said two picowells.
16. The plate of claim 11, wherein the interwell area between two said
picowells is less then about 0.06 the sum of the areas of said two picowells.
17. The plate of claim 11, wherein a rim of a said picowell is substantially
knife-edged.
18. The plate of claim 1, wherein said plurality of picowells comprises
picowells having dimensions of less than about 200 microns.
19. The plate of claim 18, wherein the dimensions of said picowells are less
than about 100 microns.
20. The plate of claim 1 8, wherein the dimensions of said picowells are less
than about 50 microns,
21. The plate of claim 18, wherein the dimensions of said picowells are less
than about 25 microns.
22. The plate of claim 18, wherein the dimensions of said picowells are less
than about 10 microns.
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23. The plate of claim 1, wherein said plurality of picowells comprises
picowells configured to hold no more than one living cell of a certain size.
24. The plate of claim 1, wherein said plurality of picowells comprises
picowells configured to hold no more than a predetermined number of living cells of a
certain size.
25. The plate of claim 1, wherein picowells of said plurality of picowells
comprise enclosures of dimensions such that substantially an entire cell of a certain size
is containable within a said enclosure, each said enclosure having an opening, said
opening defined by a first cross section of a size allowing passage of a cell of a certain
size.
26. The plate of claim 25, wherein the volume of a said enclosure is less than
about 1 x 1(T 11 liter.
27. The plate of claim 25, wherein the volume of a said enclosure is less than
about 1 x 10" 12 liter.
28. The plate of claim 25, wherein the volume of a said enclosure is less than
about 1 x 10" 13 liter.
29. The plate of claim 25, wherein the volume of a said enclosure is less than
about 1 x 10" 14 liter.
30. The plate of claim 25, wherein the volume of a said enclosure is less than
about 1 x 10" 15 liter.
31. The plate of claim 25, wherein the area of a said first cross section is less
than about 40000 micron 2 .
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32. The plate of claim 25, wherein the area of a said first cross section is less
than about 10000 micron 2 .
33. The plate of claim 25, wherein the area of a said first cross section is less
than about 2500 micron 2 .
34. The plate of claim 25, wherein the area of a said first cross section is less
than about 625 micron 2 .
35. The plate of claim 25, wherein the area of a said first cross section is less
than about 100 micron 2 .
36. The plate of claim 25, a said enclosure having dimensions so as to
contain no more than one cell of said certain size at any one time.
37. The plate of claim 25, a said enclosure having dimensions so as to
contain no more than a predetermined number of cells of said certain size at any one
time.
38. The plate of claim 1, said plurality of picowells comprising picowells,
wherein all picowells of the plate are substantially identical in size.
39. The plate of claim 1, wherein a first said well includes a first said
plurality of picowells and a second said well includes a second said plurality of
picowells, wherein said first plurality of picowells and said second plurality of
picowells are substantially different.
40. The plate of claim 39, wherein the size of picowells of said first plurality
of picowells and the size of picowells of said second plurality of picowells are
substantially different.
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41. The plate of claim 1, comprising a material selected from the group
consisting of ceramics, elastomers, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, polystyrene, polyurethane, polyvinyl chloride, rubber, silicon,
silicon oxide and silicon rubber.
42. The plate of claim 1, said bottom surface comprising a material selected
from the group consisting of ceramics, elastomers, epoxies, glasses, glass-ceramics,
metals, plastics, polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol,
polymers, polymethyl methacrylate, polystyrene, polyurethane, polyvinyl chloride,
rubber, silicon, silicon oxide and silicon rubber.
43. The plate of claim 1, wherein the walls of wells of said plurality of wells
are integrally formed with said bottom surface.
44. The plate of claim 1, further comprising at least one distinct well-wall
component attached to said bottom surface.
45. The plate of claim 44, said distinct well-wall component comprising a
material selected from the group consisting of ceramics, elastomers, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate, polystyrene,
polyurethane, polyvinyl chloride, rubber, silicon, silicon oxide and silicon rubber.
46. The plate of claim 1 , wherein said plurality of picowells are integrally
formed with said bottom surface.
47. The plate of claim 1, further comprising at least one distinct picowell-
bearing component bearing said plurality of picowells attached to said bottom surface of
said one well.
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48. The method of claim 47, wherein said pi co well-bearing component is a
carrier comprising a plurality of picowells disposed on a surface.
49. The plate of claim 47, said distinct picowell-bearing component made of
a material selected from the group consisting of reversibly deformable materials and
irreversibly deformable materials.
50. The plate of claim 47, wherein said distinct picowell-bearing component
is made of a material selected from the group consisting of waxes, hydrocarbon waxes,
crystalline waxes, paraffins, ceramics, elastomers, epoxies, glasses, glass-ceramics,
metals, plastics, polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol,
polymers, polymethyl methacrylate, polystyrene, polyurethane, polyvinyl chloride,
rubber, silicon, silicon oxide and silicon rubber.
51. The plate of claim 1, further comprising at least one distinct picowell-
bearing component bearing said plurality of picowells resting within said one well.
52. The method of claim 51, wherein said picowell-bearing component is a
carrier comprising a plurality of picowells disposed on a surface.
53. The plate of claim 51, wherein said distinct picowell-bearing component
is made of a material selected from the group consisting of reversibly deformable
materials and irreversibly deformable materials.
54. The plate of claim 51, wherein said distinct picowell-bearing component
is made of a material selected from the group consisting of gels, hydrogels, waxes,
hydrocarbon waxes, crystalline waxes, paraffins, ceramics, elastomers, epoxies, glasses,
glass-ceramics, metals, plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate, polystyrene,
polyurethane, polyvinyl chloride, rubber, silicon, silicon oxide and silicon rubber.
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55. The plate of claim 51, wherein said picowell-bearing component
comprises a gel.
56. The device of claim 55, wherein said gel is substantially transparent.
57. The device of claim 55, wherein said gel is a hydrogel.
58. The device of claim 55, wherein the water content of said gel is greater
than about 80% by weight.
59. The device of claim 55, wherein the water content of said gel is greater
than about 92% by weight.
60. The device of claim 55, wherein the water content of said gel is greater
than about 95% by weight.
61. The device of claim 55, wherein the water content of said gel is greater
than about 97% by weight.
62. The device of claim 55, wherein the water content of said gel is greater
than about 98% by weight.
63. The device of claim 55, wherein said gel is made of a material selected
from the group consisting of agar gels, agarose gels, gelatins, low melting temperature
agarose gels, alginate gels, room-temperature Ca 2+ -induced alginate gels and
polysaccharide gels.
64. The device of claim 55, wherein said gel comprises an active entity.
65. The device of claim 64, wherein said active entity is selected from the
group consisting of antibodies, antigens, biological materials, chemical materials,
chromatogenic compounds, drugs, enzymes, fluorescent probes, immunogenes,
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indicators, ligands, nucleic acids, nutrients, peptides, physiological media, proteins,
receptors, selective toxins and toxins.
66. The plate of claim 1, said plurality of picowells comprising picowells,
said picowells having a bottom surface made of a first material and borders made of a
second material, said second material being substantially different from said first
material.
67. The plate of claim 66, wherein said first material is substantially the
material from which said bottom of said well is made.
68. The plate of claim 66, wherein said bottom surface of said picowell is
substantially said bottom surface of said well.
69. The plate of claim 66, wherein said second material is fixed photoresist
material.
70. The plate of claim 1, wherein said plurality of picowells comprises
picowells having an inside surface configured to delay adhesion of living cells thereto.
71. The plate of claim 70, wherein said inside surface comprises an
adhesion-delaying material .
72. The plate of claim 70, wherein said inside surface is coated with said
adhesion-delaying material.
73. The plate of claim 1, said plurality of picowells comprising picowells,
the bottom of said picowells substantially having an index of refraction similar to that of
water.
74.
1.4.
The plate of claim 73, wherein said index of refraction is less than about
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75. The plate of claim 73, wherein said index of refraction is less than about
1.38.
76. The plate of claim 73, wherein said index of refraction is less than about
1.36.
77. The plate of claim 73, wherein said index of refraction is less than about
1.35.
78. The plate of claim 73, wherein said index of refraction is less than about
1.34.
79. The plate of claim 1, said plurality of picowells comprising picowells,
said picowells having an inner surface coated with a layer of a material.
80. The plate of claim 79, said material selected from the group consisting of
gels, hydrogels, polydimethylsiloxane, elastomers, polymerized para-xylylene
molecules, polymerized derivatives of para-xylylene molecules, rubber and silicon
rubber.
81. The plate of claim 1, further comprising a gel cover covering said
plurality of picowells.
82. The plate of claim 1, wherein said plurality of picowells covers
substantially the entire said bottom surface of said well.
83. The plate of claim 1, further comprising at least one additional feature
functionally associated with said plurality of picowells.
84. The plate of claim 83, wherein said additional feature comprises a
microfluidic feature functionally associated with said plurality of picowells.
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85. The plate of claim 84, wherein said feature is selected from the group
consisting of channels, coupling elements, drains, fluid channels, fluid reservoirs, input
ports, membranes, microreactors, microvalves, output ports, passages, plumbing routes,
protruberances, pumps, transport channels and valves.
86. The plate of claim 83, wherein said feature is selected from the group
consisting of light sources, magnetizable elements, optical components, optical fibers,
optical filters, protuberances, fiducial points and walls.
87. The plate of claim 1, further comprising a cover slip, said cover slip and
said plurality of picowells configured so as to allow said cover slip to rest above said
plurality of picowells substantially in parallel to said bottom surface.
88. A method of making a multiwell plate of claim 1, comprising:
(a) contacting a precursor material with a template including a negative of
features of the plate so as to create said features in said precursor material, said features
including said plurality of picowells;
(b) fixing said features in said precursor material so as to fashion an incipient
plate; and
(c) processing said incipient plate so as to fashion the plate.
89. The method of claim 88, wherein said template comprises a material
selected from the groups consisting of reversibly deformable materials, irreversibly
deformable materials, ceramics, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, paraffins, polystyrene, polyurethanes, polyvinyl chloride,
silicon, silicon oxide, silicon rubbers and wax.
90. The method of claim 88, wherein said fixing comprises a method
selected from the group consisting of heating said precursor material, cooling said
precursor material, polymerizing said precursor material, cross-linking said precursor
material, curing said precursor material, irradiating said precursor material, illuminating
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said precursor material, gelling said precursor material, exposing said precursor material
to a fixative and waiting a period of time.
91. The method of claim 88, said features further comprising at least one
feature selected from the group of features consisting of channels, coupling elements,
drains, fluid channels, fluid reservoirs, input ports, microreactors, microvalves,
passages, optical components, optical filters, output ports, plumbing routes,
protruberances, pumps, transport channels, valves, walls, patrial walls and fiducial
points.
92. The method of claim 88, said features further comprising said plurality of
wells.
93. The method of claim 88, further comprising:
(d) prior to (a), placing said precursor material in a well of a multiwell plate.
94. The method of claim 88, further comprising:
(d) subsequent to (b), attaching walls of said plurality of wells to said incipient
plate.
95. The method of claim 94, wherein said attaching comprises using an
adhesive to attach said walls to said incipient plate.
96. The method of claim 94, wherein said attaching comprises using a
surface treatment to attach said walls to said incipient plate.
97. The method of claim 96, wherein said surface treatment is a plasma
treatment.
98. The method of claim 88, wherein said precursor material includes a
irreversibly deformable precursor material and said fixing said features comprises
separating said template from said precursor material.
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99. The method of claim 98, wherein said irreversibly deformable precursor
material is selected from the group consisting of wax, paraffins, plastics and polymers.
100. The method of claim 88, wherein said precursor material comprises an
reversibly deformable precursor material.
101. The method of claim 100, wherein said reversibly deformable precursor
material is selected from the group consisting of gellable fluids, polymerizable
materials, powders, fluids and thermoplastic materials.
102. The method of claim 101, wherein said reversibly deformable precursor
material includes a thermoplastic material at plastic temperature and wherein said fixing
said features comprises cooling said thermoplastic material.
103. The method of claim 101, wherein said reversibly deformable precursor
material includes a polymerizable material and wherein said fixing said features
comprises polymerizing said polymerizable material.
104. The method of claim 103, wherein said polymerizable material is
selected from the group consisting of monomer solutions, crosslinkable polymers,
vulcanizable polymers, polymerizable fluids and thermosetting resins.
105. The method of claim 104, wherein said polymerizable material includes
a polydimethylsiloxane precursor mixture and said fixing said features comprises
polymerizing said polydimethylsiloxane precursor mixture so as to produce
polydimethylsiloxane.
106. The method of claim 104, wherein said polymerizable material includes
urethane and said fixing said features comprises polymerizing said urethane to produce
polyurethane.
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107. The method of claim 101, wherein said reversibly deformable precursor
material includes a gellable fluid and wherein said fixing said features comprises gelling
said gellable fluid.
108. The method of claim 107, wherein said gelling said gellable fluid
comprises an action selected from the group consisting of heating said gellable fluid,
cooling said gellable fluid, irradiating said gellable fluid, illuminating said gellable
fluid, contacting said gellable fluid with a gelling reagent and waiting a period of time
for said gellable fluid to gel.
109. The method of claim 107, wherein said gellable fluid comprises a
solution including a material selected from the group consisting of agars, agaroses,
gelatins, low melting temperature agaroses, alginates, proteins, protein polysaccharides,
Ca 2+ -inducable alginates and polysaccharides.
1 1 0. The method of claim 107, wherein said gellable fluid includes an alginate
solution and said gelling said gellable fluid comprises contacting said gellable fluid with
a gelling reagent.
111. The method of claim 1 10, wherein said gelling reagent comprises Ca 2+
ions.
112. The method of claim 107, wherein said gellable fluid includes a low
melting temperature agarose solution and said gelling said gellable fluid comprises
cooling said gellable fluid.
113. The method of claim 88, wherein said processing said incipient plate
comprises coating an inside surface of picowells of said plurality of picowells with a
layer of a coating material.
114. The method of claim 113, wherein said coating said inside surface
comprises:
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(i) applying a precursor fluid with said inside surface of said picowells;
and
(ii) solidifying said precursor fluid so as to form said layer of said
coating material.
115. The method of claim 114, wherein said solidifying comprises a method
selected from the group consisting of heating said precursor fluid, cooling said
precursor fluid, polymerizing said precursor fluid, cross-linking said precursor fluid,
curing said precursor fluid, irradiating said precursor fluid, illuminating said precursor
fluid, gelling said precursor fluid, exposing said precursor fluid to a fixative and waiting
a period of time.
116. The method of claim 113, wherein said coating said inside surface
comprises:
(i) depositing a vapor of said coating material onto said inside surface of
said picowells thereby forming said layer of said coating material.
117. The method of claim 113, wherein said coating said inside surface
comprises:
(i) depositing a vapor of a coating precursor material onto said inside
surface of said picowells; and
(ii) solidifying said coating precursor material thereby forming said layer
of said coating material.
118. The method of claim 117, wherein said solidifying said coating precursor
material comprises a method selected from heating said coating precursor material,
cooling said coating precursor material, polymerizing said coating precursor material,
cross-linking said coating precursor material, curing said coating precursor material,
irradiating said coating precursor material, illuminating said coating precursor material,
gelling said coating precursor material, exposing said coating precursor material to a
fixative and waiting a period of time.
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119. The method of claim 117, wherein said vapor is a vapor of para-xylylene
molecules or derivatives thereof and said layer of said coating material comprises a
layer of polymerized said para-xylylene molecules or derivatives thereof
120. A method of making a multiwell plate of claim 1, comprising:
(a) placing a photoresist material on a precursor plate; and
(b) fixing a plurality of picowells in said photoresist material.
121. The method of claim 120 wherein said fixing comprises irradiating said
photoresist material through a mask.
122. The method of claim 120, wherein said precursor plate comprises a
material selected from the group consisting of ceramics, epoxies, glasses, glass-
ceramics, metals, plastics, polycarbonates, polydimethylsiloxane, polymers,
polyethylenterephtalate glycol, polymethyl methacrylate, polystyrene, polyurethanes,
polyvinyl chloride, silicon and silicon oxide.
123. The method of claim 120, wherein said precursor plate comprises a
multiwell plate.
124. The method of claim 120, further comprising:
(c) subsequent to (b), attaching walls of wells to said precursor plate.
125. The method of claim 120, further comprising:
(c) subsequent to (b), coating an inside surface of picowells of said plurality of
picowells with a layer of a coating material.
126. A method of making a multiwell plate of claim 1, comprising placing a
picowell-bearing component on a precursor plate.
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127. The method of claim 126, further comprising attaching said picowell-
bearing component to said precursor plate
128. The method of claim 127, wherein said attaching comprises using an
adhesive to attach said picowell-bearing component to said precursor plate.
129. The method of claim 127, wherein said attaching comprises a surface
treatment to attach said picowell-bearing component to said precursor plate.
130. The method of claim 129, wherein said surface treatment is a plasma
treatment.
131. The method of claim 126, wherein said precursor plate comprises a
multiwell plate.
132. The method of claim 126, further comprising attaching walls of wells to
said precursor plate.
133. The method of claim 126, wherein said picowell-bearing component is a
carrier comprising a plurality of picowells disposed on a surface.
134. The method of claim 126, further comprising, subsequent to said
attaching of said picowell-bearing component, coating an inside surface of picowells of
said picowell-bearing component with a layer of a coating material.
135. A device comprising an array of living cells held in a non-fluid matrix,
said matrix configured to maintain cell viability.
136. The device of claim 135, wherein said living cells are physically held in
pockets in said matrix.
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137. The device of claim 135, wherein there is substantially no bond between
said living cells and said matrix.
138. The device of claim 135, wherein said array is substantially planar
having an upper surface and a lower surface.
139. The device of claim 138, wherein at least one said surfaces is transparent
to at least one wavelength of light.
140. The device of claim 139, wherein a said transparent surface is said upper
surface.
141. The device of claim 139, wherein a said transparent surface is said lower
surface.
142. The device of claim 139, wherein said at least one wavelength of light is
in the ultraviolet spectrum.
143. The device of claim 139, wherein said at least one wavelength of light is
in the visible spectrum.
144. The device of claim 139, wherein said at least one wavelength of light is
in the infrared spectrum.
145. The device of claim 135, said matrix comprising a material having an
index of refraction substantially similar to that of water.
146. The device of claim 145, said matrix comprising a material having an
index of refraction less than about 1 .4.
147. The device of claim 145, said matrix comprising a material having an
index of refraction less than about 1.38.
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148. The device of claim 145, said matrix comprising a material having an
index of refraction less than about 1.36.
149. The device of claim 145, said matrix comprising a material having an
index of refraction less than about 1 .35.
150. The device of claim 145, said matrix comprising a material having an
index of refraction less than about 1 .34.
151. The device of claim 135, said matrix configured to substantially delay
proliferation of living cells held therein.
152. The device of claim 135, said matrix made of a material comprising a
gel.
153. The device of claim 135, said matrix comprising an active entity.
1 54. The device of claim 1 53, said active entity comprising an indicator.
155. The method of claim 153, said active entity comprising an indicator, said
indicator configured to indicate a cell response to a stimulus.
156. The method of claim 155, wherein said cell response is a release of a
second active entity.
1 57. A method for handling living cells, comprising:
(a) providing an ordered array of living cells immobilized in a non-fluid matrix,
said matrix configured to maintain cell viability;
(b) contacting said living cells with a stimulus; and
(c) detecting a response of said cells to said stimulus.
158. The method of claim 157, said matrix comprising an active entity
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159. The method of claim 158, said active entity comprising an indicator.
160. The method of claim 1 59, said active entity comprising an indicator, said
indicator configured to indicate a cell response to a stimulus.
161. The method of claim 160, wherein said cell response is a release of a
second active entity.
162. The method of claim 157, wherein said detecting comprises contacting
an active entity with said matrix.
163. The method of claim 162, further comprising waiting a period of time so
as to allow said active entity to reach proximity with said cells.
164. The method of claim 162, said active entity comprising an indicator.
165. The method of claim 162, said active entity comprising an indicator, said
indicator configured to indicate a cell response to a stimulus.
166. The method of claim 165, wherein said cell response is a release of a
second active entity.
167. The method of claim 157, wherein said detecting comprises detecting
emitted light.
168. The method of claim 157, wherein said detecting comprises detecting
light.
169. A method of producing an ordered array of living cells in a non-fluid
matrix, comprising:
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(a) providing a multiwell plate provided with a plurality of wells, said multiwell
plate including a plurality of picowells at the bottom of at least one said well, said
plurality of picowells including picowells;
(b) placing a suspension of a plurality of living cells in a gellable fluid in said at
least one well;
(c) causing said living cells to settle into said picowells so as to be held in
respective picowells; and
(d) gelling said gellable fluid so as to make a gel cover, trapping said living cells
between said picowells and said gel cover.
170. The method of claim 169, wherein said picowells are made of a material
comprising a gel.
171. The method of claim 169, wherein said causing said cells to settle
comprises applying a force to said cells.
172. The method of claim 171, wherein said force is a force resulting from the
impact of photons.
173. The method of claim 171, wherein said force is a force resulting from a
pressure wave.
174. The method of claim 171 , wherein said force is gravitation.
1 75. The method of claim 171, wherein said force is a centrifugal force.
1 76. The method of claim 1 69, wherein
(e) prior to (d), ensuring that substantially each picowell holds no more than one
living cell.
177. The method of claim 169, wherein said gelling said gellable fluid
comprises an action selected from the group consisting of heating said gellable fluid,
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cooling said gellable fluid, irradiating said gellable fluid, illuminating said gellable
fluid, contacting said gellable fluid with a gelling reagent and waiting a period of time
for said gellable fluid to gel.
178. The method of claim 169, wherein said gellable fluid is selected so as to
form a hydrogel upon said gelling.
179. The method of claim 169, wherein said gellable fluid is selected from
solutions containing a material selected from the group consisting of agars, agaroses,
gelatins, low melting temperature agaroses, alginates, room-temperature Ca 2+ -inducable
alginates and polysaccharides.
180. The method of claim 179, wherein said gellable fluid is an alginate and
said gelling said gellable fluid comprises contacting said gellable fluid with a gelling
reagent.
181. The method of claim 179, wherein said gellable fluid is a low melting
temperature agarose and said gelling said gellable fluid comprises cooling said gellable
fluid.
182. The method of claim 169, wherein said gellable fluid comprises an active
entity.
183. The method of claim 169, said active entity comprising an indicator.
184. The method of claim 183, said active entity comprising an indicator, said
indicator configured to indicate a cell response to a stimulus.
185. The method of claim 184, wherein said cell response is a release of a
second active entity.
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It . ■
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FIG. 5A
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to
WO 2005/007796
PCT/IL2004/000661
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WO 2005/007796 PCT/IL2004/000661
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WO 2005/007796
PCT/IL2004/000661
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FIG. 12C
FIG. 12D
WO 2005/007796
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